Lignans from the Roots and Rhizomes of Dysosma versipellis and Their Cytotoxic Activities

One new dibenzyltyrolactone lignan dysoslignan A (1), three new arylnaphthalide lignans dysoslignan B–C (2–4), along with fourteen known metabolites (5–18), were isolated from the roots and rhizomes of Dysosma versipellis. Their structures and stereochemistry were determined from analysis of NMR spectroscopic and circular dichroism (CD) data. Compound 2 represents the first report of naturally occurring arylnaphthalide lignan triglycoside. The cytotoxic activities of all isolated compounds were evaluated against A-549 and SMMC-7721 cell lines. Compounds 7–10 and 14–16 were more toxic than cisplatin in two tumor cell lines. This investigation clarifies the potential effective substance basis of D. versipellis in tumor treatment.


Introduction
Arylnaphthalide lignans have received much attention due to their potent antiviral, antineoplastic, anti-inflammatory, and immunosuppressive properties [1]. The representative effective component (such as podophyllotoxin) has been the subject of extensive research on new antiviral and antineoplastic drugs. Podophyllotoxin tincture is used clinically to treat condyloma acuminatum. Podophyllotoxin derivatives, for instance etoposide and teniposide, are the frontline chemotherapeutic drugs against various cancers. Since remote times, plants containing podophyllotoxin and its analogues have been used by diverse nationalities as laxatives and for the treatment of gonorrhea, tuberculosis, menstrual disorders, psoriasis, dropsy, cough, syphilis and venereal warts [2,3]. So a medicinal plant rich in arylnaphthalide lignans is an important source of natural anticancer agents.
All isolated compounds were evaluated for their in vitro cytotoxic activities against the A-549 and SMMC-7721 cell lines using the MTS assay [22] with cisplatin and paclitaxel as positive controls, and the IC 50 values are summarized in Table 3. Compounds 7-12 and 14-17 showed more potent cytotoxicities against the SMMC-7721 cell line than the A549 cell line. Compounds 7-10 and 14-16 exhibited more potent activities than cisplatin in two tumor cell lines. Compound 14 showed the highest cytotoxicity against the A-549 and SMMC-7721 cell lines, with IC 50 values of 0.130 and 0.0088 µM, respectively. The glycosylation of 7 -hydroxy group strongly reduced the activity; for example, comparing 16 to 15, 17 to 14, and 2, 5, and 6 to 7. The cis-fusion compounds (6, 7 and 8) between the tetraline and lactone were more cytotoxic than those corresponding trans-fusion analogues (16, 15,  and 14). Compounds 7, 15 and 8, 14 containing a non-aromatized ring C exhibited more cytotoxic activity than aromatized compounds 9 and 10, indicating that the non-aromatized ring C played an important role in the cytotoxicity against A-549 and SMMC-7721 cells lines. The methylenedioxy-bearing compound (9) was found to be more potent than the ring A-opened analogue (3). The preliminary structure-activity relationship investigation suggested that the trans-fusion between the tetraline and lactone, non-aromatized ring C, and a methylenedioxy at ring A, were structurally required for maintaining cytotoxicity for related podophyllotoxin analogues.

Plant Material
The roots and rhizomes of D. versipellis were collected in Qingzhen, Guizhou Province, China, in July 2019, and identified by Prof. Cheng-Ming Dong at School of Pharmacy, Henan University of Chinese Medicine, where a voucher specimen (DV 20190706) was deposited.

Extraction and Isolation
The powered roots and rhizomes of D. versipellis (40 kg) were refluxed with 95% EtOH (v/v 120 L × 3, 1.5 h each) and 50% EtOH (v/v 120 L × 1, 1.5 h each) at 95°C, respectively. The filtrate was evaporated under reduced pressure to give a dark brown residue (5.4 kg

Acid Hydrolysis and Sugar Determination
The absolute configurations of the galatose and glucose moieties were determined by the previously reported method [19]. Compounds 1 (1.0 mg) and 2 (1.0 mg) were dissolved in 1.0 mL of 2M HCl, and then hydrolyzed at 90 • C for 3 h. The HCl in the reaction mixture was removed under reduced pressure. The remaining reaction mixture was extracted with CH 2 Cl 2 . The water layers were directly analyzed by HPLC [column: Asahipak NH 2 P-50 4E (4.6 mm × 250 mm); mobile phase: CH 3

Cytotoxicity Asssay
By the previously reported MTS method [22], the cytotoxic activities of compounds 1-18 were evaluated against human lung cancer A-549, hepatocellular carcinoma SMMC-7721 cell lines. The cells were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) at 37°C under 5% CO 2 in a humidified atmosphere. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). To be brief, 100 µL of cells were seeded into each well in a 96-well cell culture plate in advance. After 24 h, various concentrations of all test compounds were added. After the incubation for 48 h, MTS (20 µL) was added to each well, and the incubation continued for 4 h at 37°C. The optical density at 492 nm was determined using a 96-well microtiter plate reader. The IC 50 values were calculated by the Reed-Muench method. Statistical analysis were performed by SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). All experiments were performed in triplicate.