Studies on Cytotoxic Constituents from the Leaves of Elaeagnus oldhamii Maxim. in Non-Small Cell Lung Cancer A549 Cells

Elaeagnus oldhamii Maxim. is a commonly used traditional herbal medicine. In Taiwan the leaves of E. oldhamii Maxim. are mainly used for treating lung disorders. Twenty five compounds were isolated from the leaves of E. oldhamii Maxim. in the present study. These included oleanolic acid (1), 3-O-(Z)-coumaroyl oleanolic acid (2), 3-O-(E)-coumaroyl oleanolic acid (3), 3-O-caffeoyl oleanolic acid (4), ursolic acid (5), 3-O-(Z)-coumaroyl ursolic acid (6), 3-O-(E)-coumaroyl ursolic acid (7), 3-O-caffeoyl ursolic acid (8), 3β, 13β-dihydroxyolean-11-en-28-oic acid (9), 3β, 13β-dihydroxyurs-11-en-28-oic acid (10), uvaol (11), betulin (12), lupeol (13), kaempferol (14), aromadendrin (15), epigallocatechin (16), cis-tiliroside (17), trans-tiliroside (18), isoamericanol B (19), trans-p-coumaric acid (20), protocatechuic acid (21), salicylic acid (22), trans-ferulic acid (23), syringic acid (24) and 3-O-methylgallic acid (25). Of the 25 isolated compounds, 21 compounds were identified for the first time in E. oldhamii Maxim. These included compounds 1, 4, 5 and 8–25. These 25 compounds were evaluated for their inhibitory activity against the growth of non-small cell lung cancer A549 cells by the MTT assay, and the corresponding structure-activity relationships were discussed. Among these 25 compounds, compound 6 displayed the best activity against the A549 cell line in vitro (CC50 = 8.56 ± 0.57 μg/mL, at 48 h of MTT asssay). Furthermore, compound 2, 4, 8 and 18 exhibited in vitro cytotoxicity against the A549 cell line with the CC50 values of less than 20 μg/mL at 48 h of MTT asssay. These five compounds 2, 4, 6, 8 and 18 exhibited better cytotoxic activity compared with cisplatin (positive control, CC50 value of 14.87 ± 1.94 μg/mL, at 48 h of MTT asssay). The result suggested that the five compounds might be responsible for its clinical anti-lung cancer effect.

Lung cancer is one of the cancers which commonly causes deaths all over the world. In addition, lung cancer is the first most lethal cancer type and the third rate of occurrence cancer in Taiwan, respectively [28,29]. Traditionally, the leaves of E. oldhamii Maxim. were mainly used for treating lung disorders in Taiwan, such as coughs, asthma and pulmonary abscesses. Based on our interest in the anti-lung cancer activity of this plant, EOM (methanol extract of E. oldhamii Maxim.) and three partitioned fractions of the methanol extract of E. oldhamii Maxim., including EOE (EtOAc-soluble fraction), EOB (BuOH-soluble fraction) and EOW (water-soluble fraction) had been evaluated for their inhibitory activity against the growth of non-small cell lung cancer A549 cells by MTT assays in the present study. Non-small cell lung cancer (NSCLC) accounts for 85% of all cases of lung cancer, and the A549 cells line is a kind of non-small cell lung cancer cell which was often applied to evaluate cytotoxic activity in vitro [30,31]. The results showed that EtOAc-soluble fraction exhibited the best activity against the A549 cell line in vitro (CC 50
Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases, and A549 cell line is one kind of non-small cell lung cancer cells which was used to evaluate cytotoxic activity in vitro [30,31]. Therefore, we chose the A549 cell line to evaluate cytotoxic activity of these 25 compounds. In this study, four triterpenoid compounds (compounds 2, 4, 6 and 8) and one flavanoid compound (compound 18) isolated from the crude extract displayed excellent cytotoxic activity against the A549 cell line with CC 50 values of less than 20 μg/mL.
Oleanolic acid and ursolic acid exhibited good cytotoxic activity against the A549 cell line in previous studies [53][54][55]. However, compound 2, 4, 6 and 8 were oleanolic acid and ursolic acid 3-O-derivatives, and these four compounds showed better cytotoxic activity than oleanolic acid (compound 1) and ursolic acid (compound 5) in the present study. The CC 50 (Table 4). Furthermore, regard to function groups of these four compounds, compound 2 and 6 were oleanolic acid and ursolic acid 3-O-linkage to cis-coumaryl, and compound 4 and 8 were 3-O-linkage to caffeoyl. In addition, oleanolic acid and ursolic acid 3-O-linkage to cis-coumaroyl (compounds 2 and 6) exhibited better cytotoxic activity than 3-O-linkage to trans-coumaroyl (compounds 3 and 7). The CC 50 value of compound 2 and 3 were 9.46 ± 1.50 and 65.21 ± 7.15, and compounds 6 and 7 were 8.56 ± 0.57 and 79.59 ± 5.77 repectively (Table 4). Therefore, we believe that 3-O-linkage to cis-coumaroyl and caffeoyl of oleanolic acid and ursolic acid were two active functional groups responsible for the cytotoxic activity against the growth of non-small cell lung cancer A549 cells according to the MTT assay.
Kaempferol was investigated for its cytotoxic activity against the A549 cell line in a previous study [56]. Compound 17 was a kaempferol derivative and exhibited better cytotoxic activity than kaempferol (compound 14) in present study. The CC 50 value of compound 14 was 113.48 ± 5.32, and that of compound 17 was 18.82 ± 3.64 at 48 h of MTT assay, respectively (Table 4). Furthermore, with regard to the functional groups of compound 17, it has a kaempferol 3-O-linkage to D-glucose and a 6''-O-linkage to a cis-coumaroyl moiety. In addition, the kaempferol 6''-O-linkage to a cis-coumaroyl group (compound 17) exhibited better cytotoxic activity than a 6''-O-linkage to a trans-coumaroyl group (compound 18). The CC 50 value of compound 17 was 18.82 ± 3.64, and that of compound 18 was 144.74 ± 5.37 at 48 h MTT assay, respectively (Table 4). Therefore, we also believe that in this case the 3-O-linkage to D-glucose and 6''-O-linkage to the cis-coumaroyl were the active functional groups that provide better cytotoxic activity against the growth of non-small cell lung cancer A549 cells according to the MTT assay.
In chloroform-d; c In methanol-d 4 ; d In pyridine-d 5 .

Plant Material
The leaves of E. oldhamii Maxim. as described in Flora of Taiwan [1] were purchased from the Jin-Shun Shen herbal garden (Nantou, Taiwan) in October 2012. The plant materials were also identified by Yuan-Shiun Chang, Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung, Taiwan. A plant specimen has been deposited in the Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources with voucher specimen number CMU-CMR-2013-EO-1.

Extraction and Isolation
The materials were totally dried in air under dark. Dried leaves of E. oldhamii Maxim. (10.0 kg) were cut into small pieces and soaked in 70 L of methanol for 7 days. The extraction was repeated three times. The extracts were combined and filtered through filter paper and concentrated under reduced pressure to give a dried extract (801.0 g). The dried extract was suspended in H 2 O (2 L) and extracted with ethyl acetate (2 L, five times). The resulting ethyl acetate extract was concentrated to yield 302.5 g of a brown-green thick oil. The fraction was further purified on 2.2 kg of silica gel with particle size 0.063-0.200 mm on a column with 10 cm internal diameter and 50 cm length using a gradient of increasing polarity from total n-hexane to total ethyl acetate as mobile phase and was separated into 20 fractions. Fraction 1 (0.52 g) was isolated from the 100% n-hexane mobile phase. Fraction 2 (12.28 g) was isolated from the 1:19 n-hexane/ethyl acetate mobile phase. Fraction 3 (18.95 g) was isolated from the 1:9 n-hexane/ethyl acetate mobile phase. Fractions 4 (10.28 g), 5 (11.41 g), 6 (10.55 g), 7 (15.20 g) and 8 (8.85 g) were isolated from the 8:2 n-hexane/ethyl acetate mobile phase. Fractions 9 (16.69 g) and 10 (13.44 g) were isolated from the 7:3 n-hexane/ethyl acetate mobile phase. Fractions 11 (19.18 g), 12 (11.74 g), 13 (7.84 g) and 14 (11.40 g) were isolated from the 5:5 n-hexane/ethyl acetate mobile phase. Fractions 15 (6.02 g), 16 (14.38 g) and 17 (6.01 g) were isolated from the 3:7 n-hexane/ethyl acetate mobile phase. Fractions 18 (19.91 g), 19 (12.57 g) and 20 (6.13 g) were isolated from the 100% ethyl acetate mobile phase. The ethyl acetate extract remaining on the column was eluted by methanol (28.92 g). The weight of the 20 fractions and the fraction which was eluted by methanol was 261.94 g, so the recovery rate was 86.59%.

Evaluation of Cell Proliferation by MTT Assay
The number of viable A549 cells after GA treatment was evaluated by the MTT (3-[4,5-methylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay. In brief, A549 cells (3 × 10 4 cells/well) were seeded in a 96-well plate and kept overnight for attachment. The next day the medium was replaced with fresh medium with various concentrations of EOM extract, indicated fraction (EOE, EOB, and EOW), cisplatin (positive control) and 25 compounds which were isolated from the leaves of E. oldhamii Maxim. (5-500 µg/mL) and cells were allowed to grow for 24 and 48 h. Four hours before completion of incubation, 10 µL of MTT (10 mg/mL) was added in each well. After completing the incubation, 100 µL of solubilization buffer (10% SDS with 0.01 N HCl) was added to each well and incubated overnight at room temperature. The optical density (OD) was measured at 450 nm using a multiscan microplate reader. Data were showed means ± SD from three independent experiments, 50% cytotoxic concentration (CC 50 ) yielding 50% toxic effect was determined via a computer program (provided by John Spouge, National Center for Biotechnology Information, National Institutes of Health).

Conclusions
Twenty five compounds were isolated in the present study, including thirteen triterpenoids, five flavonoids, one lignanoid and six benzanoids compounds. Furthermore, twenty one of these compounds, including compounds 1, 4, 5 and 8-25 were obtained from E. oldhamii Maxim. for the first time. Furthermore, we evaluated the inhibitory activity of these compounds against the growth of non-small cell lung cancer A549 cells by MTT assay in this study. Eleven of these 25 compounds, including compounds 2-4, 6-10, 13, 18 and 19 were investigated by the MTT assay for the first time regarding their cytotoxic activity in non-small cell lung cancer A549 cells. The result indicated that the five compounds, including four triterpenoids (compounds 2, 4, 6 and 8) and one flavanoid (compound 18) present in the crude extract exhibited good cytotoxic activity against the A549 cell line with CC 50 values lower than 20 μg/mL. Furthermore, in present study these five compounds exhibited better cytotoxic activity compared to cisplatin (positive control, Table 4).
Regarding the structure-activity relationships of the triterpenoid compounds 2, 4, 6 and 8, a 3-O-linkage to the cis-coumaroyl and caffeoyl moieties of oleanolic acid and ursolic acid were two active functional groups that provided better cytotoxic activity against the growth of the non-small cell lung cancer A549 cells according to the MTT assays. In addition, regarding the structure-activity relationships of the flavanoid compound 18, a 3-O-linkage to D-glucose and a 6''-O-linkage to cis-coumaroyl were the active functional groups providing better cytotoxic activity against the growth of non-small cell lung cancer A549 cells in the MTT assay. These five compounds 2, 4, 6, 8 and 18 may lead to the development of anti-lung cancer drugs although further studies should be performed to reveal the mechanisms of action of the active compounds found in leaves of E. oldhamii Maxim.