Anti-Proliferative Activity of Triterpenoids and Sterols Isolated from Alstonia scholaris against Non-Small-Cell Lung Carcinoma Cells

(1) Background: In China and South Asia, Alstonia scholaris (Apocynaceae) is an important medicinal plant that has been historically used in traditional ethnopharmacy to treat infectious diseases. Although various pharmacological activities have been reported, the anti-lung cancer components of A. scholaris have not yet been identified. The objective of this study is to evaluate the active components of the leaf extract of A. scholaris, and assess the anti-proliferation effects of isolated compounds against non-small-cell lung carcinoma cells; (2) Methods: NMR was used to identify the chemical constitutes isolated from the leaf extract of A. scholaris. The anti-proliferative activity of compounds against non-small-cell lung carcinoma cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; (3) Results: Eight triterpenoids and five sterols were isolated from the hexane portion of A. scholaris, and structurally identified as: (1) ursolic acid, (2) oleanolic acid, (3) betulinic acid, (4) betulin, (5) 2β,3β,28-lup-20(29)-ene-triol, (6) lupeol, (7) β-amyrin, (8) α-amyrin, (9) poriferasterol, (10) epicampesterol, (11) β-sitosterol, (12) 6β-hydroxy-4-stigmasten-3-one, and (13) ergosta-7,22-diene-3β,5α,6β-triol. Compound 5 was isolated from a plant source for the first time. In addition, compounds 9, 10, 12, and 13 were also isolated from A. scholaris for the first time. Ursolic acid, betulinic acid, betulin, and 2β,3β,28-lup-20(29)-ene-triol showed anti-proliferative activity against NSCLC, with IC50 of 39.8, 40.1, 240.5 and 172.6 μM, respectively.; (4) Conclusion: These findings reflect that pentacyclic triterpenoids are the anti-lung cancer chemicals in A. scholaris. The ability of ursolic acid, betulinic acid, betulin, and 2β,3β,28-lup-20(29)-ene-triol to inhibit the proliferative activity of NSCLC can constitute a valuable group of therapeutic agents in the future.


Introduction
In the past few decades, non-small-cell lung cancer (NSCLC), one of the most commonly diagnosed malignancies, has been shown to be the leading cause of cancer-related mortality all over the world. In all lung cancer cases, 75% to 80% have been identified as non-small-cell lung cancer, while only 15% to 25% is small cell lung cancer (SCLC). It is noted that conventional treatment of either form of lung cancer is fairly ineffective [1]. Thus, the development of new therapeutic strategies against NSCLC is urgently needed. Previous studies have demonstrated that extracts from some herbal medicines have anti-lung cancer potential and can inhibit lung cancer cell proliferation [2][3][4][5]. Recently, many of the chemotherapeutic agents are medicinal plants or are derived from medicinal plants. Therefore, attention has been paid to investigate the natural, active ingredients from medicinal plants against lung cancer cell.
The Alstonia scholaris, belonging to the family Apocynaceae, is widely distributed in the tropical regions of Africa and Asia [6]. It is a tropical evergreen tree native to South and Southeast Asia, and is called blackboard tree, or milkwood pine, commonly. Traditionally, the leaves of A. scholaris have been used in "Dai" ethnopharmacy to treat chronic respiratory diseases in China [7]. In Africa, Australia, India, Malaysia, the Philippines, and Thailand, A. scholaris are also used in traditional medicinal systems [7]. The extracts of A. scholaris possess a wide spectrum of pharmacological activities; as a result, the chemical constituents of A. scholaris, especially the alkaloids, have been extensively investigated [8][9][10][11][12][13]. The extracts of A. scholaris have been observed to possess anti-diabetic [14], antiinflammatory [15], anti-tussive, anti-asthmatic, and expectorant activities [16]. Recently, the potential of A. scholaris on antimicrobial activity has been screened, and the potent chemical constitutes and their exact effective concentration have also been identified [17]. These findings reflect that the pleiotropic effects of ursolic acid against methicillin-resistant Staphylococcus aureus (MRSA) make it a promising antibacterial agent in pharmaceutical research [18]. Although the pharmacological usage of A. scholaris has been greatly investigated, the anti-proliferative activity against NSCLC is not clear. Therefore, the aim of this study was to further investigate the anti-proliferative constitutes from the leaf extracts of A. scholaris against NSCLC. It is suggested that these compounds might be a valuable group of therapeutic agents in NSCLC treatment in the future.

Antiproliferation Activity of Triterpenoids and Steriols against NSCLC
To evaluate the anti-proliferative activities of isolated triterpenoids ( Figure 2A) and sterols ( Figure 2B) on NSCLC cells, A549 cells were treated with various concentrations of isolated compounds for 48 h. The cell viability was evaluated using the MTT assay. As shown in Figure 2A, the exposure of A549 cells to compounds 1, 3, 4 and 5 decreased cellular viability in a dose-dependent

Antiproliferation Activity of Triterpenoids and Steriols against NSCLC
To evaluate the anti-proliferative activities of isolated triterpenoids ( Figure 2A) and sterols ( Figure 2B) on NSCLC cells, A549 cells were treated with various concentrations of isolated compounds for 48 h. The cell viability was evaluated using the MTT assay. As shown in Figure 2A, the exposure of A549 cells to compounds 1, 3, 4 and 5 decreased cellular viability in a dose-dependent manner.
In the treatment of sterols, only compound 11 showed an inhibitory effect on NSCLC cells, with a 20% decrease in cell viability. Interesting, compounds 9 and 10 showed no inhibiting effect on A549 cells, but did show an increasing proliferation effect ( Figure 2B). These results showed that only triterpenoids exhibited efficient anti-proliferative effects on NSCLC cells in an A. scholaris leaf extract. manner. In the treatment of sterols, only compound 11 showed an inhibitory effect on NSCLC cells, with a 20% decrease in cell viability. Interesting, compounds 9 and 10 showed no inhibiting effect on A549 cells, but did show an increasing proliferation effect ( Figure 2B). These results showed that only triterpenoids exhibited efficient anti-proliferative effects on NSCLC cells in an A. scholaris leaf extract.

Discussion
Triterpenoids are a group of structurally diverse metabolites that are often used as pharmaceuticals with various biological activities. Triterpenoids exist abundantly in Alstonia spp. and their proposed bioactivities include anti-HIV, anti-microbial, allelopathy, anti-tumor, and anti-cancer activities [17,[30][31][32][33]. In addition, the pharmacological activities of A. scholaris, particularly anti-lung cancer activity, have not yet been fully explained. Previously, the main triterpenoids in leaves of A. scholaris were identified by HPLC and LC/MS/MS [31]. Seven triterpenoid peaks were identified as cylicodiscic acid (7.7%), betulin (5.8%), betulinic acid (5.4%), oleanolic acid (15.1%), ursolic acid (23.6%), cycloeucalenol (10.3%), and α-amyrin acetate (6.5%), respectively. They found that the portion of triterpenoids showed a high anti-proliferative activity in A549 cells with IC 50 values of 9.3 µg/mL. Several papers reported that ursolic acid possesses strong anti-cancer activity against several cancers of the prostate, breast, lung, pancreas, and bladder [34][35][36][37]. Ursolic acid had been isolated from R. formosanum, an endemic species distributed widely in Taiwan [38]. Way et al. focused on the antineoplastic effect of ursolic acid on NSCLC cells, and found that ursolic acid activated AMP-activated protein kinase (AMPK), and then inhibited the mTOR pathway, which controls protein synthesis and cell growth. These findings suggested that ursolic acid is a potent anti-cancer agent. In this study, we have investigated the chemical constituents and anti-proliferative activity of A. scholaris against NSCLC cells. We found that the major components with anti-proliferative activity in the leaves of A. scholaris were ursolic acid and betulinic acid. Oleanolic acid did not possess any anti-proliferative activity against A549 cells in this study. Moreover, compound 5 (2β,3β,28-lup-20(29)-ene-triol) also showed anti-proliferative activity against A549 cells. Our data suggest that not only ursolic acid, but also betulinic acid, is a potent anti-cancer agent. Previously studies have demonstrated that betulinic acid has anti-proliferative properties in vitro and in vivo [39,40]. Betulinic acid was able to trigger the mitochondrial pathway of apoptosis to induce apoptotic cell death in cancer cells [41,42]. In mice, pharmacokinetic studies demonstrated that betulinic acid was well absorbed and distributed within the melanoma xenografts [43]. In addition, normal cells and tissue are relatively resistant to betulinic acid, pointing to a therapeutic usage [44]. Moreover, betulinic acid is being developed by a large network of clinical trial groups supported by the U.S. National Cancer Institute [45]. Therefore, it is tempting to propose that A. scholaris could be developed as an anti-cancer agent for NSCLC.
Although sterols isolated in this study exhibited no cytotoxic effects on NSCLC cells, the ability of sterols in clinical trials to block cholesterol absorption sites in the human intestine. It is worth investigating whether sterols could help reduce cholesterol absorption in humans, especially these first isolated sterols from A. scholaris, including poriferasterol, epicampesterol, 6β-hydroxy-4-stigmasten-3-one, and ergosta-7,22-diene-3β,5α,6β-triol. In conclusion, the ability of ursolic acid, betulinic acid, betulin, and 2β,3β,28-lup-20(29)-ene-triol to inhibit the proliferative activity of NSCLC can constitute a valuable group of therapeutic agents in the future.

Isolation and Identification of Triterpenoids and Sterols
As shown in Figure 3, the anti-proliferative constitutes of the most effective fractions in the hexane portion (fraction Hex-4 to Hex-7) were isolated by using column chromatography to obtain 13 pure compounds.   Table 1.

Anti-Proliferative Activity
Antiproliferation activity was determined against A549 cells (human lung adenocarcinoma cell line) using the MTT assay (Promega, Fitchburg, WI, USA). Briefly, the A549 cell line was cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin). This assay is based on the

Anti-Proliferative Activity
Antiproliferation activity was determined against A549 cells (human lung adenocarcinoma cell line) using the MTT assay (Promega, Fitchburg, WI, USA). Briefly, the A549 cell line was cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin). This assay is based on the cleavage of the MTT to purple formazan crystals by metabolically active cells. MTT assay was done as described previously [38]. Briefly, the A549 cells were inoculated into a 96-well culture plate (1 × 10 4 cells/well) and treated with tested compounds in different concentrations at 37 • C for 48 h. After removing the medium from each well, 100 µL of MTT (500 µg/mL) was added to each well, and the plate was incubated at 37 • C for 1 h. When purple precipitate was clearly visible under the microscope, 80 µL of DMSO was added to each well. The plate was incubated in the dark for 1 h at room temperature. The spectrophotometric absorbance of the samples was detected by using an ELISA reader (SpectraMax M5e, Molecular Devices LLC, Sunnyvale, CA, USA) at 570 nm. The cell viability was calculated as the percentage of cell survival after the treatment. All measurements were performed in triplicate.

Conflicts of Interest:
The authors declare no conflict of interest.