Two New C21 Steroidal Glycosides from the Roots of Cynanchum paniculatum

Abstract Two new C21 steroidal glycosides, paniculatumosides H and I, together with four known ones were isolated from the roots of Cynanchum paniculatum (Bge.) Kitag. Their structures were identified by spectroscopic methods including extensive 1D and 2D NMR techniques. All compounds were subjected to detect the anti-tobacco mosaic virus (TMV) activities and their cytotoxities against three human tumor cell lines (SMMC-7721, MDA-MB-231 and A549). The results showed that compounds 1 and 5 exhibited potent protective activities against TMV, while 2, 4 and 6 had moderate effects on the SMMC-7721 cancer cells viability. Graphical Abstract


Introduction
Cynanchum paniculatum (Bunge) Kitag is a vivacious herb broadly distributed in China, Japan and Korea, whose dried roots have been used as a Chinese herbal medicine for the treatment of rheumatic arthralgia, epigastric pain, toothache, lumbago, traumatic injuries, and eczema [1][2][3]. In the previous work, it has been confirmed that C. paniculatum contains C 21 steroidal glycosides [4,5]. Pregnanes and their glycosides have shown many aspect activities, such as antitumor, antifungal, antiviral and cytotoxic activities [6,7]. Our previous works found that pregnanes and their glycosides could inhibit tobacco mosaic virus through suppressing the expression of viral subgenomic RNA but without affecting the accumulation of viral genomic RNA [6]. Therefore, in order to find the structurally unique natural products in C. paniculatum and explore their biological activities, we investigated the dichloromethane extract of C. paniculatum, and two new steroidal glycosides paniculatumosides H (1) and I (2), together with four known ones glaucogenin C (3) [8], cynatratoside A (4) [8], cynapanoside A (5) [9] and 1 3 neocynapanogenin F 3-O-β-d-oleandropyranoside (6) [10] were isolated. Furthermore, we tested the anti-TMV activities and cytotoxicity of these compounds (Fig. 1).  (Fig. 2). The main difference was an extra sugar unit present at 1, which could be further verified by the glycosidation shifts at C-2 (− 3.8), C-3 (+ 4.2) and C-4 (− 2.2).

Anti-TMV Activities
All compounds were tested for anti-TMV activity using the half-leaf method. The results revealed that compounds 1 and 5 exhibited protective activities at concentration of 200 μM, with the antiviral inhibition rates of 62.7% and 59.5%, respectively. Ningnanmycin, which was used as a positive control, showed inhibition rates of 57.3%.

Cytotoxic Activities
In order to evaluate whether these compounds have any

Plant Material
The roots of C. paniculatum were purchased from a medicinal market (Kunming luosiwan Chinese herbal medicine market) in August 2017 and identified by prof. Hua Peng of Kumming Institute of Botany, Chinese Acdemy of Sciences (CAS).

Extraction and Isolation
The roots of C. paniculatum (100.0 kg) were powdered and extracted three times with MeOH at room temperature to afford 7.2 kg of crude extract. The extract was partitioned between CH 2 Cl 2 and aqueous solution portions which yielded 4.4 kg crude CH 2 Cl 2 extract. This extract was subjected to normal-phase silica gel column chromatography eluted with a gradient of petroleum ether-acetone (from 1:0 to 1:2) and CH 2 Cl 2 -MeOH (10:1-0:1) to obtain eight major fractions (Fr.

Anti-TMV Assays
The half-leaf method was used to evaluate the anti-TMV activities as literatures reported [14]. Ningnanmycin, a Chinese commercial product for plant disease, obtained from Heilongjiang Qiang'er Biochemical Technology Development Company, was administered as a positive control.

Cytotoxicity Assay
The cytotoxicity of each compound on three cultured human cancer cell lines was tested by MTT assay. The cell lines used were SMMC-7721 (human hepatoma cells), MDA-MB-231 (triple-negative breast cancer cells), A549 (human lung cancer cell). Cell growth inhibition assay was performed as reported literatures [15]. Cisplatin was used as a positive control.