Anti-Proliferative Effect of Triterpenoidal Glycosides from the Roots of Anemone vitifolia through a Pro-Apoptotic Way

A cytotoxicity-guided phytochemical investigation of Anemone vitifolia roots led to the isolation of six oleanane saponins (1–6), which were reported from the species for the first time. Their structures were determined by comparing its MS and NMR data with those in literature. Compounds 1–4 showed significant inhibitory effects on the proliferation of hepatocellular carcinoma HepG2 cells with IC50 values ranging from 2.0 to 8.5 μM, compared to positive control methotrexate with IC50 value of 15.8 μM. Flow cytometry analysis revealed that compounds 1–4 exerted anti-proliferative effects through a pro-apoptotic way of hepatocellular carcinoma cells.


Introduction
Anemone L. is a big genus in the family of Ranunculaceae, which consists of 150 species across the world [1]. As the major and characteristic constituents in this genus, oleanolic acid and hederagenin triterpenoid saponin showed anti-tumor, anti-inflammatory, and analgesic activities, etc. [2][3][4][5][6]. A. vitifolia Buch-Ham is one of the most representative medicinal plants, which is mainly distributed in northwestern and southwestern China, northern Burma, Bhutan, Sikkim, Nepal, and northern India [1]. Long-term practice and traditional use has proved its significant activity on the treatment of rheumatoid joint pain, enteritis, and diarrhea, in addition to its use in natural pesticides [7][8][9]. However, no phytochemical and pharmacological activity study of this botanical specimen has yet been performed.
A cytotoxicity-guided fractionation on the roots of A. vitifolia was conducted, which led to the isolation and identification of six triterpenoidal glycosides (1-6). The compounds showed significant inhibitory effects on cell proliferation of hepatocellular carcinoma with IC 50 values ranging from 2.5 to 12.0 µM, compared to the positive control of methotrexate. Flow cytometry analysis revealed that compounds 1-4 remarkably induced apoptosis of HepG2 cells. Here in, this work documents the isolation, cytotoxic evaluation and apoptotic analysis of these constituents from A. vitifolia.

Structural Identification of Compounds 1-6
Although A. vitifolia is traditionally used for the treatment of rheumatoid arthritis, no in vitro anti-inflammatory activity was observed in our previous study of this specimen. Inspired by Anemone saponins with significant antitumor activities reported in References [10][11][12][13], A. vitifolia was chemically investigated by a bioassay-guided separation against HepG2 cell lines in this study, which led to the discovery of the cytotoxic butanol soluble extract, fraction ABB, and six compounds (1-6). The chemical structures of the abovementioned compounds are shown in Figure 1.
Clematichinenoside A (5) (Table S2). A comparison of the NMR data with that of 3 suggested its structural similarity, except for an additional glycosylation at C-28. All of the above data is in good agreement with that previously reported [22], thus the structure of 5 was determined.

Flow Cytometry Analysis
Apoptosis is an important physiological mechanism of cell death. Inhibition of normal cell apoptosis in the body could led to proliferative diseases, such as tumors and autoimmune diseases. Therefore, the modulation of apoptosis may be a feasible means for the prevention and treatment of these diseases, which is an effective way for the discovery of leading compounds [23][24][25][26]. Flow cytometry analysis with Annexin V-FITC/propidium iodide (PI) is widely used to detect apoptosis. Therefore, the effects of 1-4 on the apoptosis of HepG2 cells were examined using flow cytometry analysis. As shown in Figure 3, these compounds induced apoptosis of HepG2 cells, especially at high concentrations. Moreover, most of the staining cells were in the Q4 field of flow cytometry chart after treatment of 1-4 for 48 h, suggesting that 1-4 induced apoptosis of HepG2 cells mainly at the early stage. Taken together, these saponins exerted anti-proliferative effect through triggering apoptosis of hepatocellular carcinoma cells.

Flow Cytometry Analysis
Apoptosis is an important physiological mechanism of cell death. Inhibition of normal cell apoptosis in the body could led to proliferative diseases, such as tumors and autoimmune diseases. Therefore, the modulation of apoptosis may be a feasible means for the prevention and treatment of these diseases, which is an effective way for the discovery of leading compounds [23][24][25][26]. Flow cytometry analysis with Annexin V-FITC/propidium iodide (PI) is widely used to detect apoptosis. Therefore, the effects of 1-4 on the apoptosis of HepG2 cells were examined using flow cytometry analysis. As shown in Figure 3, these compounds induced apoptosis of HepG2 cells, especially at high concentrations. Moreover, most of the staining cells were in the Q 4 field of flow cytometry chart after treatment of 1-4 for 48 h, suggesting that 1-4 induced apoptosis of HepG2 cells mainly at the early stage. Taken together, these saponins exerted anti-proliferative effect through triggering apoptosis of hepatocellular carcinoma cells.

Flow Cytometry Analysis
Apoptosis is an important physiological mechanism of cell death. Inhibition of normal cell apoptosis in the body could led to proliferative diseases, such as tumors and autoimmune diseases. Therefore, the modulation of apoptosis may be a feasible means for the prevention and treatment of these diseases, which is an effective way for the discovery of leading compounds [23][24][25][26]. Flow cytometry analysis with Annexin V-FITC/propidium iodide (PI) is widely used to detect apoptosis. Therefore, the effects of 1-4 on the apoptosis of HepG2 cells were examined using flow cytometry analysis. As shown in Figure 3, these compounds induced apoptosis of HepG2 cells, especially at high concentrations. Moreover, most of the staining cells were in the Q4 field of flow cytometry chart after treatment of 1-4 for 48 h, suggesting that 1-4 induced apoptosis of HepG2 cells mainly at the early stage. Taken together, these saponins exerted anti-proliferative effect through triggering apoptosis of hepatocellular carcinoma cells.

Preliminary Structure-Activity Relationship Analysis
A brief structure-activity relationship (SAR) analysis ( Figure 4) of compounds 1-6 inferred that the presence of free C-28 carboxyl functionality is crucial to the cytotoxicity against HepG2 cells. This is the reason why compounds 1-4 were significantly active rather than 5 or 6. SAR analysis of 1-4 indicated that the ribose as the outer sugar might play an important role, and the xylose as the inner sugar also has considerable impact on the activity. However, this is only a preliminary SAR result, and more samples are required for a substantial conclusion.

Preliminary Structure-Activity Relationship Analysis
A brief structure-activity relationship (SAR) analysis ( Figure 4) of compounds 1-6 inferred that the presence of free C-28 carboxyl functionality is crucial to the cytotoxicity against HepG2 cells. This is the reason why compounds 1-4 were significantly active rather than 5 or 6. SAR analysis of 1-4 indicated that the ribose as the outer sugar might play an important role, and the xylose as the inner sugar also has considerable impact on the activity. However, this is only a preliminary SAR result, and more samples are required for a substantial conclusion.

Preliminary Structure-Activity Relationship Analysis
A brief structure-activity relationship (SAR) analysis ( Figure 4) of compounds 1-6 inferred that the presence of free C-28 carboxyl functionality is crucial to the cytotoxicity against HepG2 cells. This is the reason why compounds 1-4 were significantly active rather than 5 or 6. SAR analysis of 1-4 indicated that the ribose as the outer sugar might play an important role, and the xylose as the inner sugar also has considerable impact on the activity. However, this is only a preliminary SAR result, and more samples are required for a substantial conclusion.

Cytotoxicity Assay
The extracts, fractions and isolated compounds were dissolved in DMSO (Sigma, St. Louis, MO, USA, WSBB5403V, purity >99% by HPLC) as stock solutions. Before each bioassay, all of the stock solutions were diluted with DMEM (cellgro, 1 × with 4.5 g/L glucose, L-glutamine & sodium pyruvate, Manassas, VA, USA) to give final concentrations of 0.4, 4 and 40 µM containing less than 0.1% DMSO. HepG2 cells were seeded in 96-well plates at 3.5 × 10 3 cells/well and incubated for 24 h. After that, the extracts, fractions and isolated compounds were added as described above for another 48-h incubation. In the meantime, methotrexate was used as a positive control, and during each bioassay 100 µL MTT (0.5 mg/mL) was added into each well for 4 h of incubation. Following that, the supernatant in each well was thrown and 150 µL DMSO was added. The plates were swiftly shaken to fully dissolve crystals, and then transferred to a microplate reader to measure the optical density at a wavelength of 570 nm.

Flow Cytometry Analysis
HepG2 cells were seeded in 12-well plates at a density of 4 × 10 4 cells/well and were treated with 1-4. Drugs with four concentrations were added to each plate, and each concentration was added to three wells for repetition. After treatment of drugs for 48 h, cells were collected and a reagent was added in Annexin V-FITC apoptosis detection kit (BD Pharmingen), according to the manufacturer's instructions. Furthermore, early apoptotic cells were prominently stained by Annexin V-FITC, while late apoptotic cells were stained by PI (propidium iodide).

Data Analysis
The data are expressed as the mean ± the standard error of the mean (SEM), using Graph Pad Prism 5 and the Statistical Package for the Social Sciences (SPSS) 20.0 software. The differences of means of the measured parameters were compared by using one-way analysis of variance (ANOVA). The p-values < 0.01 were regarded as significant.

Conclusions
A cytotoxicity-guided fractionation on the roots of Anemone vitifolia led to the isolation of six oleanane saponins. Four of them (1-4) showed significant inhibition on the cell proliferation of hepatocellular carcinoma HepG2 cells with IC 50 values of 2.0-8.5 µM, compared to positive control methotrexate with an IC 50 value of 15.8 µM. Flow cytometry analyses revealed that these saponins exert the in vitro cytotoxic effect by remarkably inducing apoptosis of HepG2 cells, and a preliminary SAR analysis suggests that the free carboxyl in the molecules plays a vital role in this kind of biological activity. This paper represents the phytochemical investigation of the species, as well as the inhibitory effect of compounds 2, 4, and 6 on the proliferation of HepG2 cells, for the first time.
The present findings will not only enrich the discovery of cytotoxic leads for liver disease drug development, but also provide a reference for oleanane saponins research, especially for its SAR analysis.
Supplementary Materials: Supplementary materials are available online. HRESIMS and 1 H and 13 C NMR spectroscopic spectra of compounds 1-6 and separation flow.