Steroidal Saponins from Vernonia amygdalina Del. and Their Biological Activity

In the present study, four new steroidal saponins, namely vernoniamyoside A–D (1–4), together with the two known steroidal saponins vernoamyoside D (5) and vernonioside B2 (6) were isolated from the ethanol extract of leaves of the African medicinal plant Vernonia amygdalina Del. (Asteraceae). Their structures were demonstrated by spectral analyses along with 1D and 2D nuclear magnetic resonance (NMR) techniques and mass spectrometry (MS). The cytotoxicity of the compounds was also tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method on the cell lines Hela, MCF-7, BT-549 and MDA-MB-231. Vernoniamyoside A, vernoniamyoside B, and vernonioside B2 showed cytotoxicity towards BT-549 cell lines. Vernoniamyoside C, vernoniamyoside D and vernoamyoside D showed different levels of cytotoxic activities.


Introduction
Vernonia amygdalina Del. from the family Asteraceae is distributed throughout tropical Africa, especially in West Africa. It has received considerable scientific interest due to the observation that adult chimpanzees with malaria returned to normal activity after chewing the extract of the bitter juice of this species [1]. Over the years, several studies on the chemical components of this species, including flavonoids [2], sesquiterpene lactones [3], steroidal saponins [4][5][6], and fatty acids [7], have been performed. Previous studies have indicated different bioactivities of this species, including anti-inflammation [8], anti-malaria [9], anti-obesity [10,11], antioxidant [12,13], anti-tumor [14], and other activities [15]. Water and chloroform extracts of V. amygdalina (VA) interfere with the DNA synthesis of MCF-7 cells in breast cancer, affecting the activity of ERKs in vitro and inhibiting human breast cancer cells [16,17]. Via the MTT method, it has been verified that MCF-7 cells are inhibited in vitro and the DNA synthesis of BT-549 cells in breast cancer is disturbed by the addition of VA extract [18,19]. Moreover, three active fractions extracted from ethanol extracts of V. amygdalina have an inhibiting effect on tumor cells, the essential component being steroidal saponins [14]. So far, a certain amount of steroidal saponins has been obtained from V. amygdalina; however, studies on the anti-tumor activity are rare. It is therefore especially important to investigate the separation and biological activity of steroidal saponins in V. amygdalina.
In the present study, four new steroidal saponins, vernoniamyoside A-D (1-4) along with two known steroidal saponins vernoamyoside D (5) and vernonioside B 2 (6) were obtained. It is expected that these steroidal saponins also demonstrate anti-tumor activity, especially anti-breast cancer activity. Therefore, all compounds were evaluated for their cytotoxicity toward human Hela, MCF-7, BT-549, and MDA-MB-231 cell lines by means of the MTT method. The isolation, structure identification, and biological activities of the aforementioned compounds are described.
Compound 3 was obtained as a white powder, and its molecular formula C 35 16), which has correlation with δ C 48.7 (C-14) in the HMBC spectrum. The correlations between H-17 (δ H 2.41) and the carbon signal at δ C 74.3 in the HMBC spectrum confirm that the δ C 74.3 is connected to C-16. The 1 H NMR and 13 C NMR signals (see Table 1) of compound 3 indicated that it is a ∆ 7, 9 (11) stigmastane-type steroid derivative with the same skeleton as vernoamyoside D [6]. According to the HMBC, the signal at δ H 7.49 has a correlation with δ C 56.6 (C-17), 173.3 (C-21), 82.8 (C-23), while δ H 2.41 (H-17) and 5.24 (H-23) have correlations with δ C 133.9. Therefore, there is a double bond between C-20 (δ C 133.9) and C-22 (δ C 146.8). Thus, the planar structure of compound 3 could be determined. Compared to the 1 H NMR and 13 C NMR signals, the side chain is the same as in vernonioside A4 [20]. The significant nuclear overhauser effect (NOE) correlations between H-16 (δ H 4.40) and Me-18 (δ H 0.30) suggested that OH-16 has an β-configuration. Therefore, compound 3 was determined and named vernoniamyoside C (see Figures 1 and 5).    Table 1) of compound 3 indicated that it is a Δ 7, 9 (11) stigmastane-type steroid derivative with the same skeleton as vernoamyoside D [6]. According to the HMBC, the signal at δH 7.49 has a correlation with δC 56.6 (C-17), 173.3 (C-21), 82.8 (C-23), while δH 2.41 (H-17) and 5.24 (H-23) have correlations with δC 133.9. Therefore, there is a double bond between C-20 (δC 133.9) and C-22 (δC 146.8). Thus, the planar structure of compound 3 could be determined. Compared to the 1 H NMR and 13 C NMR signals, the side chain is the same as in vernonioside A4 [20]. The significant nuclear overhauser effect (NOE) correlations between H-16 (δH 4.40) and Me-18 (δH 0.30) suggested that OH-16 has an β-configuration. Therefore, compound 3 was determined and named vernoniamyoside C. (see Figure 1    Compound 3 was obtained as a white powder, and its molecular formula C35H50O11, determined by HR-ESI-MS at m/z 669.3250 [M + Na] + (calcd for C35H50NaO11, 669.3206), has 11 degrees of unsaturation. The proton signal is at δH 4.40 (H-16), which has correlation with δC 48.7 (C-14) in the HMBC spectrum. The correlations between H-17 (δH 2.41) and the carbon signal at δC 74.3 in the HMBC spectrum confirm that the δC 74.3 is connected to C-16. The 1 H NMR and 13 C NMR signals (see Table 1) of compound 3 indicated that it is a Δ 7, 9 (11) stigmastane-type steroid derivative with the same skeleton as vernoamyoside D [6]. According to the HMBC, the signal at δH 7.49 has a correlation with δC 56.6 (C-17), 173.3 (C-21), 82.8 (C-23), while δH 2.41 (H-17) and 5.24 (H-23) have correlations with δC 133.9. Therefore, there is a double bond between C-20 (δC 133.9) and C-22 (δC 146.8). Thus, the planar structure of compound 3 could be determined. Compared to the 1 H NMR and 13 C NMR signals, the side chain is the same as in vernonioside A4 [20]. The significant nuclear overhauser effect (NOE) correlations between H-16 (δH 4.40) and Me-18 (δH 0.30) suggested that OH-16 has an β-configuration. Therefore, compound 3 was determined and named vernoniamyoside C. (see Figure 1 Table 1) of compound 2, compound 4 is also a ∆ 7, 9 (11) stigmastane-type steroid derivative with a glycoside. As for the side chain, δ H 5.22 (H-23) has correlations with δ C 135.7 in the HMBC spectrum. Compound 4 had similar 1 H, 13 C NMR, and NOESY data when compared with compound 3. Thus, compound 4 was determined and named vernoniamyoside D (see Figures 1 and 6).  Table 1) of compound 2, compound 4 is also a Δ 7, 9 (11) stigmastane-type steroid derivative with a glycoside. As for the side chain, δH 5.22 (H-23) has correlations with δC 135.7 in the HMBC spectrum. Compound 4 had similar 1 H, 13 C NMR, and NOESY data when compared with compound 3. Thus, compound 4 was determined and named vernoniamyoside D (see Figure 1 and 6).

Results of the Cytotoxicity Test
According to a previous study, MCF-7 cells are inhibited in vitro, and the DNA synthesis of BT-549 cells in breast cancer is disturbed by the application of V. amygdalina extract [18,19]. A series of steroidal saponins exhibited anti-tumor activity, especially anti-breast tumor activity [14]. In this study, the cytotoxicity of compounds 1-6 was tested on BT-549, MDA-MB-231, MCF-7, and Hela cell lines. The anti-tumor activity of ∆ 7, 9 (11) stigmastane-type steroidal saponins was introduced for the first time. As seen in Table 2, the inhibition against the BT-549 cell line of compound 1 could reach up to 63.61%, while compound 2 and 6 also showed cytotoxicity towards the BT-549 cell line (inhibition = 62.17 and 51.14%). This leads us to infer that compounds 1, 2, and 6 are highly toxic towards BT-549 cell lines, while they showed a general cytotoxicity to cell lines MDA-MB-231, MCF-7, and Hela. Based on this, these compounds might play a certain role in the treatment of breast cancer. The cytotoxicity activities of compound 3, 4, and 5 showed different levels against the tested cell lines. Moreover, further studies are necessary to confirm whether these compounds are also toxic towards other tumor cell lines. Both compounds have the same sugar chains; therefore, the different activities might be due to the side chain and the nuclear parent. Furthermore, it should be highlighted that compounds 1-6 was had a different selectivity for tumor cell lines.

General Experimental Procedures
Optical rotations were measured with an Automatic polarimeter (Hackettstown, NJ, USA). UV spectra were recorded by a Shimadzu UV-2600 PC spectrophotometer (Suzhou, Jiangsu, China). IR (KBr-disks) spectra were measured using a Bruker Alpha (Karlsruhe, Germany). The HR-ESI-MS spectra were recorded by a Thermo Scientific Q Exactive Plus Orbitrap LC-MS/MS system (Waltham, MA, USA); NMR spectra were recorded by a Bruker AVANCE III 600 MHz spectrometer (Zurich, Switzerland) in CD 3 COCD 3 , with TMS as internal standard. The preparative high-performance liquid chromatography (HPLC) system consisted of an LC-6AD intelligent prep. pump (Kyoto, Japan), an SPD-20A intelligent UV/VIS detector (Kyoto, Japan), and a YMC-Park ODS-A column (5 µm, 250 × 10 mm I.D., YMC Co. Ltd., Ishikawa, Japan). Silica gel GF 254 for thin-layer chromatography (TLC) and silica gel (200-300 mesh) for column chromatography (CC) were obtained from Qingdao Marine Chemical Factory (Qingdao, Shandong, China). Sephadex LH-20 (Merck, Darmstadt, Germany) was suited for size-exclusion chromatography. The cell lines BT-549 Hela, MCF-7, and MDA-MB-231 were obtained from China Center for Type Culture Collection (Wuhan, Hubei, China); MTT were obtained from Sigma Company. All solvents were purchased from Sinopharm Chemical Reagents (Shanghai, China). Methyl alcohol used for HPLC analysis was of chromatographic grade (Sigma, St. Louis, MO, USA). All aqueous solutions were prepared with double-distilled water.

Plant Material
Dried leaves of V. amygdalina were collected in Xiamen, Fujian Province, China, and identified by Qing Chen, Assistant Professor from the School of Pharmaceutical Sciences, Xiamen University.

Extraction and Isolation
The dried leaves (7.5 kg) were extracted with 95% ethanol and concentrated under vacuum to obtain crude extract. Subsequently, the crude extract was suspended in H 2 O and partitioned in petroleum ether, dichloromethane (CH 2 Cl 2 ), ethyl acetate, and n-butanol in sequence to obtain the petroleum ether fraction (170 g), the dichloromethane fraction (224 g), the ethyl acetate fraction (80 g), and the n-butanol fraction (60 g), respectively. The dichloromethane fraction (224 g), a dark green syrup, was subjected to macroporous resin column chromatography (CC) (4 kg, D101) and eluted with gradient methyl alcohol (MeOH)-H 2 O (8:2) and MeOH. After removing the solvents under vacuum, the MeOH-H 2 O extract (110 g) was subjected to silica gel column chromatography (CC) (1 kg, 200-300 mesh) and eluted with gradient CH 2 Cl 2 -MeOH (50:1 to 1:1 v/v) and MeOH to obtain fractions A (9.8 g), B (14.2 g), C (21.7 g), D (22.2 g), E (15.7 g), F (14.7 g), and G (8.5 g) after deducting the solvents. Fraction C was re-chromatographed on a silica gel CC eluted with CH 2 Cl 2 -MeOH, using a gradient (15:1 to 5:1 v/v) to obtain five fractions (Ca-Ce). Fraction Cb was further purified by Sephadex LH-20 eluted with MeOH to obtain 32 fractions, which were pooled together using TLC to obtain the three sub-fractions Cba-Cbc. The fraction Cbb (922 mg) was separated by preparative HPLC, using a YMC-Park ODS-A column (  in 100 µL of fetal calf serum per well. After 4 h, we removed the MTT reagent and added 150 µL DMSO to each well. Doxorubicin was used as the positive control. The sample without test compounds was used as the negative control. Absorbance was measured at 490 nm in an automated microplate reader. All experiments were performed in triplicate. Inhibition was calculated via the following equation: A test sample is the test sample absorbance, A blank is the blank absorbance, A negative control is the negative control absorbance.

Conclusions
We obtained four new steroidal saponins, namely vernoniamyoside A-D (1-4), together with the two known steroidal saponins vernoamyoside D (5) and vernonioside B 2 (6) from V. amygdalina. Of these, 1, 2, and 6 showed an excellent cytotoxicity on BT-549 cell lines in the cytotoxicity activity assay (Table 2), as expected. It is worth noting that 1 and 2 were selective for different tumor cell lines.
Our results indicate that substances 1 and 2 were toxic to BT-549 cell lines. Further studies should consider the screening of more types of tumor cells.
The novelty of the saponins 1-4 is represented by the highly oxidized groups in the side chain, which may be associated with their cytotoxic expression. Saponin 1 has a strong cytotoxicity to BT-549 cell lines, including two ketones and one ester group. It is assumed that the cytotoxicity of saponin 1 may be derived from the C=O group. This group, present in the highly oxidized side chains of saponins 2-4, may provide a basis for the cytotoxicity for BT-549. However, the different inhibitory effects may be due to the relationships between the different structures and spatial configurations. The specific mechanisms and verifications offer a new direction for further experiments.
The separated steroidal saponins from the dichloromethane extraction of V. amygdalina, are mainly hypoglycemic steroidal saponins. However, it is essential to separate polysaccharide steroidal saponins, which always exist in ethyl acetate and n-butanol extraction, from V. amygdalina. In addition to the steroidal saponins, V. amygdalina contains a series of active substances such as sesquiterpene lactones and flavonoids. Therefore, the separation and identification of these chemical components should be one of the most promising trends in the study of V. amygdalina.
Our results provide a scientific basis for the use of V. amygdalina in anti-tumor research, with a potential value in the treatment of cancer.
Supplementary Materials: The following 1 H NMR, 13 C NMR, 2D-NMR, and HR-ESI-MS spectra are available as supporting data. Supplementary materials are available online.