Cytotoxicity of Aporphine, Protoberberine, and Protopine Alkaloids from Dicranostigma leptopodum (Maxim.) Fedde

Nine alkaloids with three different structural skeletons were isolated from Dicranostigma leptopodum (Maxim.) Fedde (Papaveraceae) by repeated silica gel column chromatography. Their chemical structures were identified on the basic of physicochemical and spectroscopic data. Among them, 10-O-methylhernovine (1), nantenine (2), corytuberine (3), lagesianine A (4), and dihydrocryptopine (9) were first isolated from this plant. With a series of cytotoxic tests, compounds 2, 3, and 7 displayed cytotoxicity against SMMC-7721 with IC50 values of 70.08 ± 4.63, 73.22 ± 2.35, and 27.77 ± 2.29 μM, respectively.


Introduction
It was reported that nearly three-fifths of currently used anticancer agents were obtained from natural sources [1][2][3]. Therefore, utilization of ethnopharmacology is an important channel of discovering new biologically active compounds. Dicranostigma is a genus in the poppy family Papaveraceae, which is widely distributed in highland areas, especially in Western China. The plants of Dicranostigma have been used in folk medicine for treatment of tonsillitis, hepatitis, and inflammation in China for a long time [4][5][6][7]. With development of natural product chemistry, recent researches showed that D. leptopodum had more excellent biological activities. Extractsof D. leptopodum have been reported to exhibit antimicrobial activity [7], antiviral [8], antitumor [9], and anti-liver fibrosis activity [10], and anti-inflammatory activity [11]. Several compounds including alkaloids and terpenes have been reported from D. leptopodum [5][6][7]. But further investigation is necessary to find the chemical basis of activities in this plant. This work aimed to identify the active compounds by assessing the cytotoxic activity of alkaloids isolated from whole plant of Dicranostigma leptopodum (Maxim.) Fedde on selected cell lines. And the structural evidence related to cytotoxicity is also discussed.
In order to obtain their potential pharmacological activities, compounds 1-9 were evaluated for their cytotoxicities against H1299, MCF-7, and SMMC-7721 tumor cell lines by the CCK-8 assay. The results were listed in Table 1. Compound 2, 3, and 7 showed their cytotoxicity against SMCC-7721. It was reported that promising effects of extracts Nantenine (2) Dihydrosanguinarine (7) Protopine (8) Dihydrocryptopine (9) of D. leptopodum were determined both in vitro and in vivo on antiproliferating of SMCC-7721 cells [12]. Therefore, the cytotoxicity showed in the report [12] might mainly relate with these constituents. Other compounds (1, 4-6, 8, and 9) did not show cytotoxicity against the tested cell lines. However, there were some other biological activities of these compounds reported in the literature. This was the first time that cytotoxicity of compound 1 was reported. Compound 4 has been tested in vitro against human poliovirus and was found to be active with selectivity indices >14 [13]. Compounds 5 and 6 were reported to have weak cytotoxicity, such as nontoxic to KB cells, but showed activities in inhibiting cell proliferation of hepatocellular carcinoma [14][15][16][17][18]. The result was consistent with the literature that compound 7 often showed better cytotoxicity than compound 8 against cancer cell lines such as A549, HT-29, KB, and P-388 [19][20][21].
In aporphine type alkaloids, compounds 2 and 3 showed cytotoxicities to selected cell lines. It has been reported that compound 2 has cytotoxicity against human colon cancer (HCT-116, Caco-2) and normal colon (CCD-18Co) cell lines [22]. And compound 3 was reported to show strong activities in inhibiting the T or B cell proliferation and exhibited strong analgesic effects [23]. Both compounds 3 and 6 have 10-CH 3 and 11-OH. It has been reported that aporphine alkaloids with a 10-CH 3 substitution was negative to its activity related to D2 receptor, despite the presence of the critical 11-OH [24]. In addition, it was found that cytotoxicity of compound 3 with 1-OH was stronger than compound 6 with 1-OCH 3 . It could be inferred that cytotoxicity of aporphine alkaloids with 1-OH was stronger than those with 1-OCH 3 . And the potential drug targets of these compounds in cell might related with D2 receptor [25].
From the view of structure-activity relationship [26], it could be inferred that 1-OH together with 11-OH is necessary to exhibit cytotoxicity among aporphine alkaloids. For example, compound 3, having these two hydroxyl substitutions, showed certain cytotoxicity. Indeed, other aporphine compounds above showed weaker cytotoxicity against selected cell lines in this work.

Conclusions
Among compounds 1-9, it was obviously that protoberberine-type alkaloids had stronger cytotoxicity than protopine and aporphine-type ones. From the perspective of structure-activity relationship, it was expected that both 1-OH and 11-OH groups in aporphine alkaloids might be important to exhibit cytotoxic against selected cell lines while 1-OCH 3 exhibits a negative effect to the cytotoxic. The D2 receptor in cell might be the potential drug targets of these compounds. Moreover, further study is needed to investigate the internal mechanism of alkaloids obtained from D. leptopodum. This may become potential basis for new antitumor drugs.   . Its NMR spectral data were in accord with the reported data [27].

Material and Methods
Nantenine (2). Yellow needle crystals, soluble in chloroform, exhibited a positive Dragendorff 's test. . The above data were in accord with the literature data [30].
Corydine (5). It is colorless columnar crystals. . The 1 H-and 13 C-NMR spectral data were consistent with the literature data [31].

Cytotoxicity
Assay. The cytotoxicity of compounds 1-9 was determined by the CCK-8 assay [36]. H1299 (nonsmall lung carcinoma), MCF-7 (breast cancer), and SMMC-7721 (liver cancer) were purchased from the Chinese Academy of Medical Sciences (Beijing, China). Doxorubicin (DOX, Adriamycin, Actavis Italy S.p.A., Beijing, China) was the positive control. All cells were grown and maintained in RPMI 1640 (Sigma, St. Louis, MO, USA) medium supplemented with 10% fetal bovine serum (Grand Island, NY, USA), 100 IU/mL penicillin (Flow Lab, Beijing, China), and 100 g/mL streptomycin (Flow Lab, Beijing, China) at 37 ∘ C, 5% CO 2 , and 90% humidity. Cancer cells were seeded in the growth medium (100 L) into 96-well microtiter plate (5 × 10 3 cells per each well). After 4-6 h preincubation in the incubator (Forma Series ΙΙ Water Jacket) to allow cellular attachment, various concentrations of test solution were added and cells were incubated for 36 h. At the end of the incubation, CCK-8 reagent (Cell Counting Kit-8, Dojindo, Kumamoto, Japan, 10 L) was added into each well followed by further incubation for 2 h. The optical density (OD) was measured at 450 nm using a multiscan microplate reader (Thermo, Shanghai, China) [37]. Each determination represented the average mean of six replicates. The halfmaximal growth inhibitory concentration (IC 50  (1)

Disclosure
Samples of the crude extracts and pure compounds are available from the authors.