Four new triterpene saponins from Cephalaria speciosa and their potent cytotoxic and immunomodulatory activities

Four new triterpene saponins, namely speciosides A-D (1–4) along with six known saponins were isolated from the n-butanol extract of Cephalaria speciosa. In addition to these, three new prosapogenins (2a–4a) were obtained after alkaline hydrolysis. Elucidation of the structures of the isolated compounds was carried out by 1D, 2D NMR, HR-ESI/MS and GC–MS analyses. Cytotoxic activity was investigated on A549, CCD34-Lu, MDA-MB-231, PC-3, U-87MG, HeLa, HepG-2 cells by MTT method. Additionally, the immunomodulatory effect of compounds was evaluated for macrophage polarization with/without inactivated IBV D274 antigen treatment on THP-1 cells originated macrophage cells in terms of M1 or M2. According to the cytotoxicity results, compound 1 and prosapogenin 2a exhibit significant cytotoxicity than doxorubicin by comparison. The results demonstrated that saponin molecules treated THP-1 originated macrophages were induced M1 and/or M2 polarization. Additionally, macrophage cells treated with/without IBV D274 antigen contained saponin compounds were triggered significantly M2 polarization relative to M1. Notably, monodesmosidic saponins (1 and 2a–4a) in comparison with bisdesmosidic ones (2–4) demonstrated the most effect on M2 polarization. In conclusion, the results showed that all the isolated new saponins and their prosapogenins have immunomodulatory potential on macrophage cells increasing immune response without significant cytotoxic effect on THP-1 originated macrophages.


Plant material
All methods were carried out in accordance with guidelines on good agricultural and collection practices for medicinal plants of the World Health Organization and European Medicines Agency 17,18 .The plant material was collected in accordance with relevant institutional, national and international guidelines and legislation.The appropriate permission for the collection was provided by "Republic of Turkiye Ministry of Agriculture and Forestry, General Directorate of Nature Conservation and National Parks" with the number E-21264211-288.04-6345319.Cephalaria speciosa 17 was gathered in August 2013 from 1600 m altitude hillside between Sivas-Erzincan (39°54′01'' N, 38°51′08'' E).The plant was identified by R. Suleyman Gokturk and then was archived at Akdeniz University Herbarium Research Centre with the number R.S. Gokturk 7673.The aerial parts of the plant were harvested by hand, dried at room temperature and kept away sunlight.

Monosaccharide analysis
For the investigation of the exact structure of carbohydrate units, acidic hydrolysis and GC-MS analysis were performed.3-5 mg (each) of compounds 1-4 were hydrolyzed with 5 ml 5% HCl (aq) solution under reflux for 2 h at 90 °C.And then the reaction mixture was neutralized using a 5% KOH (aq) solution.The resulting aqueous solution was extracted with 10 ml of CHCl 3 , the extraction procedure was repeated three times for the organic and aqueous layers from each extraction step.The water phase dried and was kept in a hi-vac for 24 h.It was dissolved in 2 mL of pyridine, after dissolution 2 mL of HMDS-TMCS (1:1) (hexamethyldisilazane-trimethylchlorosilane) was added and was carried out under reflux, and argon gas at 85 °C for 2 h.A silylation reaction was carried out for the standard sugar mixture, simultaneously.The monosaccharide analysis of the compounds 1-4 was performed by the GC-Mass method of Sarikahya and Kirmizigul 10 under the following conditions; column, HP-5MS (60 m × 0.25 mm ID × 0.25 µm df), flow rate, 1.0 mL/min; split ratio, 1:20; ion source temperature, 230 °C; inlet and interface temperatures, 250 °C; temperature program started at 60 °C (held for 1 min) followed by 10 °C/min at 240 °C (held for 2 min), the final temperature reached 320 °C (held for 3 min) and the EI mode Table 2. 13 C-NMR data for compounds 1-4. 13

Alkaline hydrolysis
The esteric linkages of the sugar moieties which are in bisdesmosidic triterpene glycosides (compounds 2-4) were hydrolyzed by the following method.In this method, all glycosides (3 mg each) were refluxed with 5% KOH in a water solution (pH = 12-13) at 80 °C for 1 h.The reaction mixtures were neutralized with 5% HCl in a water www.nature.com/scientificreports/solution and then concentrated to dryness 10 .The residues were extracted with n-BuOH:H 2 O (1:1) and the organic layers were analyzed by 1 H-NMR to give the pure prosapogenins (2a-4a www.nature.com/scientificreports/
After the formazan crystals were completely dissolved, the optical density (OD) was measured at 570 nm with an UV/Vis microplate spectrophotometer (Thermo Fisher Scientific, USA).All data are the results of at least three independent experiments carried out in triplicate.Data are presented as mean ± standard error of the mean (SEM) of samples.The IC 50 value was calculated using the GraphPad Prism 8 (USA) program based on the calculated percent viability values and absorbance values.The percentage of viability was determined by Eq. ( 1):

Determination of macrophage polarization by flow cytometry
To differentiate THP-1 cells into M0-THP-1, cells were seeded at an initial concentration of 1 × 10 6 cells/mL in 6-well plates (Corning, USA) and treated with 15 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma USA).Then incubate at 5% CO 2 , 37 °C for 48 h 24 .To evaluate the potential of saponins with/without infectious bronchitis virus (IBV) D274 antigen on macrophage polarization, THP-1 cells were treated with saponins at 3 µg/ mL and then incubated for 24 h with or without 4 µL of inactivated IBV D274 antigens (final titer of 1 HA).The inactive IBV D274 antigen used in this study was prepared in our previously published study 25 .Based on the results of our previous research, the saponin concentration was selected as a non-cytotoxic concentration 9 .At the end of the incubation period, the cells were collected by trypsinization and washed three times with PBS.Following, 1 µL of PE anti-human CD80 (Biolegend, CA), FITC anti-human CD163 (Biolegend, CA), and FITC anti-human CD11b (Biolegend, CA) antibodies were added into the cells pellet and incubated for 45 min at room temperature in the dark.The cells pellets underwent two PBS washes following incubation.Finally, the cells were subsequently analyzed by flow cytometry (BD Accuri, C5). 1 × 10 4 cells were counted for each sample group using flow cytometry.All data are the results of at least three independent experiments carried out in triplicate.Data are presented as mean ± standard error of the mean (SEM) of samples.The untreated stained macrophage cells with anti-human CD80, FITC anti-human CD163, and FITC anti-human CD11b were compared with the results of treated cells to obtain from M0 polarization to M1 and M2 or both.

Statistical analysis
The results of at least three independent experiments were compared with the variable groups and the control group, with mean ± standard deviation values.One-way analysis of variance (ANOVA) was performed with GraphPad Prism 8 (USA) by Dunnett's test.P < 0.05 was considered significant.

Results and discussion
Phytochemical studies on the aerial parts of the Cephalaria speciosa resulted in a total of 10 saponins, 4 of which new triterpene saponins, namely, speciosides A-D (1-4) with 3 new prosapogenins (2a-4a) (Fig. 1).A total of 6 known saponins named macranthoidin A (5) 26 elmalienoside A (6) 27 , dipsacoside B (7) 28 , decaisoside E (8) 29 , scoposide A (9 30 were also isolated, purified and structurally identified. Compound 1 was obtained as a pale yellow amorphous powder having [α] D 25 : -10.8 (c: 0.19, DMSO).IR spectrum of compound 1 showed absorption bands of functional groups which are aliphatic C-H (2923 cm −1 ), alkene -C=C-(1693 cm −1 ), carboxylic acid carbonyl C=O (1727 cm −1 ), and C-O (1268, 1048 cm −1 ) and hydroxyl groups (3347 cm −1 ).The 13 C-NMR spectrum showed that compound 1 consisted of 42 carbons (Table 2), of which 12 were identified for carbohydrate groups and 30 carbons were determined for the aglycone.The 13   Compound 2 was obtained as a white amorphous powder with specific rotation of [α] D 25 : -6.1 (c 0.33, MeOH).The IR and NMR spectroscopic features of 2 were similar to those of 1 except for the sugar units and free typical hydroxy group linked C-23 (Tables 1 and 2).Spectral analysis of the compound 2 showed that the structure consists of 54 carbons can be seen 13 C-NMR spectrum (See.Sup.Info.Fig. S 13).Aglycone part was determined as oleanoic acid based on chemical shift difference for C-23 of aglycone compared to hederagenin where compound 2 shows signal for C-23 at δ C 27.8 methyl signal (Table 2).The structure of aglycone moiety was defined based on HSQC, COSY, and HMBC spectrum analysis in a similar way as in compound 1.The 1 H and 13 C-NMR data for the aglycone part also agree with data for oleanoic acid aglycone reported in the literature 10 .Sugar carbons were defined according to COSY and HMBC correlations.For the sugar moieties, the Compound 3 was obtained as a white amorphous powder with a specific rotation of [α] D 25 -15.9 (c 0.13, MeOH).The IR and NMR data of the aglycon moiety of 3 were identical to the other new compounds (1 and 4).The C-3 oxymethine carbon and C-28 carbonyl carbon were observed at δ C 79.5 and 175.7, respectively which suggests that 3 is a bisdesmosidic triterpene saponin.The other aglycone carbon locations were determined by studies on COSY and HMBC correlations.For the sugar moieties, the 1 H-NMR spectrum of 3 displayed four anomeric proton signals at δ H 4.26 (d, J = 7.8 Hz), 5.19 (brs), 5.18 (d, J = 8.4 Hz) and 4.17 Compound 4 was obtained as a pale yellow amorphous powder with a specific rotation of [α] D 25 -11.7 (c 0.34, MeOH).Compound 4 consists of 54 carbons according to 13 C-NMR (Table 2).24 of the carbon signals were identified for carbohydrate groups and 30 carbons were determined for the aglycone and confirmed that compound 4 has a hederagenin aglycone.The C-3 oxymethine carbon and C-28 carbonyl carbon were observed at δ C 80.0 and 175.7, respectively which suggests that compound 4 is a bisdesmosidic hederagenin type triterpene saponin.Aglycone carbon locations were determined by studies on COSY and HMBC correlations.For the carbohydrate moieties, the 1 H-NMR spectrum of compound 4 showed four anomeric proton signals at δ H 4.26 (d, J = 7.2 Hz), 5.17 www.nature.com/scientificreports/I) showed the sugar linkage points to the aglycone.Furthermore, HMBC correlations between δ H 5.17 The cytotoxic effects of compounds 1-4 and prosapogenins 2a-4a, which was obtained after alkaline hydrolysis, were examined using the MTT method.The estimated IC 50 values was given in Table 3 and the percent vitality graph according to the MTT result was given in Fig. S 60-S 67 in Sup.Info.According to the results, while compounds 2-4, and 3a did not have a cytotoxic effect on THP-1-derived macrophage cells., compounds 1, 2a, and 4a (27.57± 0.52, 37.34 ± 8.17, and 35.74 ± 4.90 µM) inflicted weak cytotoxic effect when compared with doxorubicin (6.84 ± 0.18 µM).Compounds 1, 2a, and 4a demonstrated significant cytotoxicity on most cancerous cells.The mentioned compounds showed significant IC 50 on A549 and MDA-MB-231 cells as 8.59 ± 0.19 and 15.09 ± 1.02 µM for compound 1, and 20.77 ± 0.46 and 24.29 ± 2.65 µM for prosapogenin 2a, and 8.13 ± 0.01 and 8.43 ± 0.15 µM for 4a when compared with doxorubicin (40.01 ± 0.02 and 70.00 ± 0.02 µM).Compounds 2-4 and prosapogenin 3a did not show significant cytotoxicity on all the tested cell lines (Table 3).These results demonstrated that bisdesmosidic compounds (2-4) did not show any cytotoxic activity while following monodesmosidic compounds (1, 2a, 3a, and 4a) have cytotoxic effect on cancerous cell lines.In literature, as a similar study Tlili et al. 31 , who identified the biochemical profiles of the major compounds found in plant leaf extract and examined its anti-leukemic potential against acute monocytic leukemia (AML) THP-1 cells.They proved that the mTOR pathway may be involved in cell cycle inhibition and apoptosis induction caused by saponin.Furthermore, the obtained data confirm the predictions that the monodesmosidic saponins exhibit parallel cytotoxicity in our previous studies 9,11 .Furthermore, the structures of compounds 1 and 2a have the same galactose and rhamnose units attached third carbon of aglycone moieties however their aglycone structures are hederagenin and oleanane, respectively.These structural varieties may prove the activity distinctness; cytotoxic activity on A549, MDA-MB-231, and HeLa cell lines (8.59 ± 0.19, 15.09 ± 1.02, and 48.11 ± 4.56 µM) for compound 1, and A549 and MDA-MB-231 (20.77 ± 0.46 and 24.29 ± 2.65 µM) for compound 2a.These results may relate with free -CH 2 OH of hederagenin aglycone moiety of compound 1 increased the cytotoxicity.
In this study, it was also aimed to examine the potential of saponin and their prosapogenins from C. speciosa on THP-1 monocytes-originated macrophage polarization.Regarding the immune modulation potential of saponins, THP-1-originated macrophage cells were used to determine of saponin effect on macrophage polarization.The use of the THP-1 cell line as a suitable model to study the functions and responses of monocytes and macrophages, as well as the differentiation of macrophages and any potential effects of environmental stimuli is suggested 22 .Also, Genin et al. 32 suggested the THP-1 cell line for macrophage differentiation and established a novel and practical model of human macrophage polarization to study how macrophages could modulate tumor cells, specifically the tumor cells' response to chemotherapeutic agents.For this purpose, THP-1 cells were used as a suitable model to study macrophage differentiation and the potential effects of environmental stimuli.The THP-1 cell line is recommended for macrophage differentiation and for establishing a practical model of human macrophage polarization to study how macrophages could modulate tumor cells.
The Mean Fluorescent Intensity (MFI) values (Table 5) of flow cytometry results showed that the MFI of CD11b in all THP-1 originated macrophage cells is almost near 30.000 which means all of the treated groups induced M0 polarization in THP-1 macrophages (Fig. 2).According to Fig. 2 in both antigen and without antigen saponin treated cells MFI values of CD163, the marker of M2 on macrophages when compared with CD80, the marker of M1, are higher which shows the saponins potential on M2 macrophage polarization.And also, it is worthwhile to mention the MFI values of cells treated with both antigen and saponin that is significantly higher than the cells without antigen (Fig. 2a,b).According to the fact that M1/M2 describes the two major and opposing activities of macrophages 33 .In the present study all of the samples showed the similar inverse relationship between CD80 and CD163's MFI values.Based on our results, the saponins enhanced M2 polarization.In the present study, all samples showed a similar inverse relationship between the MFI values of CD80 and CD163.5).
Regarding saponins, studies suggest that they can trigger Th2 in M0 macrophages, which in turn stimulates M2 macrophage polarization 34,35 .Furthermore, treating cells with both antigens and saponins can enhance macrophage polarization, leading to an improved immune system response.Macrophages, derived from monocytes, play a critical role in inflammation, host defense, and tissue healing.M2-polarized macrophages have potential as adjuvants for anticancer therapies, and recent approaches focus on M2 polarization.According to Zhao et al. 36 demonstrated that Panax saponins promote M2 macrophage polarization so that depend on their effects of antiinflammation, saponins have important role on treatment of vascular disease.Similarly, our findings demonstrate that saponins play an effective role in M2 macrophage polarization.As mentioned before, our findings indicate that saponins take an effective role in M2 macrophage polarization so that induce wound healing effect.

Conclusion
This study evaluated the cytotoxic activities and macrophage polarization potential of four new oleanoic acid and hederagenin type triterpene saponins (1-4) named speciosides A-D and their new prosapogenins (2a-4a), respectively.The 4 new saponins and 3 prosapogenins were tested for their cytotoxicity.Consequently, 1, 2a, and 4a exhibited significant cytotoxicity against A549 cells and MDA-MB-231 cells.All isolated new saponins (1-4) and their prosapogenins (2a-4a) induced M2 macrophage polarization.These results provide valuable information for the literature on the structure, cytotoxicity, and immunomodulatory relationship of the studied saponins.Further studies should explore the activity-structure relationships to establish a model for activitytargeted synthesis and semi-synthesis.Moreover, additional research on the biological activities of these saponins for pharmaceutical and industrial applications is recommended.

Table 1 .
1H-NMR data for compounds 1-4.1HNMR data (δ) were measured in DMSO-d 6 at 600 MHz.Coupling constants (J) in Hz are given in parentheses.The assignments are based on DEPT, COSY, HMQC and HMBC experiments.nd: not detected.
C NMR data were measured in DMSO-d 6 at 150 MHz.The assignments are based on HSQC, COSY and HMBC experiments.nd: not detected.