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)23 elmalienoside A (6)24, dipsacoside B (7)25, decaisoside E (8)26, scoposide A (9)10 (3-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl oleanolic acid 28-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (10)27 were also isolated, purified and structurally identified.
Compound 1 was obtained as a pale yellow amorphous powder having [α]D25: − 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 13C-NMR spectrum showed that compound 1 consisted of 44 carbons (Table 2), of which 12 were identified for carbohydrate groups and 32 carbons were determined for the aglycone. The 13C-NMR data of 1 revealed the presence of six methyl carbon signals at δC 13.4 (C-24), 16.0 (C-25), 17.5 (C-26), 26.1 (C-27), 33.6 (C-29), 24.0 (C-30), and 1H-NMR data showed six singlet proton signals at δH 0.53, 0.83, 0.68, 1.06, 0.83, 0.84, respectively. The aglycone part of 1 was also found to consist of one carboxylic acid carbon at δC 180.8 (C-28), one ester carbonyl at δC 162.8 and methoxy at δC 40.7, oxygen-bearing methine carbon at δC 80.0 (C-3), hydroxymethyl group at δC 62.8 (C-23), and with two typical olefinic carbon signals at δC 121.1 (C-12) and 145.4 (C-13) confirmed that compound 1 has a acetlylated hederagenin aglycone. The C-28 carbonyl carbon was observed at 180.8 which suggests that 1 is a monodesmosidic triterpene saponin. The 2D-NMR correlations for the aglycone moiety were detected as follows, between δH 0.68 proton signal (H-26) and δC 32.9 (C-7), 41.8 (C-14), 47.7 (C-9) carbon signals, δH 0.53 (H-24) singlet methyl proton signal and δC 42.6 (C-4), 46.8 (C-5), 62.8 (C-23), 80.0 (C-3), 162.8 (OCOCH3) carbon signals, δH 3.05 ( H-23) hydroxymethyl proton and δC 162.8 (OCOCH3), δH 0.83 proton signal of H-25 and δC 36.4 (C-10), 38.8 (C-1), 46.8 (C-5) carbon signals, δH 0.83 proton signal of H-30 and δC 31.0 (C-20), 33.6 (C-29) carbon signals, proton of C-29 δH 0.83 and δC 24.0 (C-30) carbon signal of aglycone. Another correlation was observed between δH 1.06 proton signal of H-27 of aglycone and δC 27.8 (C-15), 39.2 (C-8), 41.8 (C-14). The carbon signal of C-13 of aglycone, δC 145.4 (C-13) gives HMBC correlation with both proton signals δH 1.05 and 1.75 of H-11. HMBC correlations were also observed between C-23 protons (δH 3.34, 3.05) and δC 80.0 (C-3) and δC13.4, δH 2.73 (H-18) and δC 122.1, 145.4 (C12-13) carbon signals, δH 5.10 (H-12) and δC 41.8 (C-14) carbon signal. In addition to these, the COSY spectrum supported these correlations. For the carbohydrate moieties, δC 103.8 and 100.2 due to the high chemical shift values identified as anomeric carbons of sugars. δC 18.3 peak in 13C-NMR was identified as a typical signal for C-6 of rhamnose. Carbon signals at δC 103.8, 74.3, 75.5, 69.0, 75.2, 60.5 and δC 100.2, 70.7, 70.9, 72.5, 68.1, 18.3 were identified as two different carbohydrate units depending on COSY and HMBC spectra. Sugar moiety where directly bonded to aglycone was determined by HMBC correlation between δC 80.0 (C-3) and δH 4.25 (anomeric proton of galactose). Rhamnose and galactose linkage point was also identified by HMBC correlation between δC 100.2 (C-1 of rhamnose) and δH 3.44 (H-2 of galactose). All other 2D-NMR correlations supported all carbon and proton values of the two carbohydrate moieties. A GC-MS analysis of 1 also confirmed the types of sugar units. Identification of L-rhamnose and D-galactose was detected for 1, giving peaks at 13.35 and 15.89 min, respectively. Based on all these evidences IUPAC name of compound 1 is 3-O-α-L-rhamopyranosyl-(1→2)-β-D-galactopyranosyl-23-Acetyloxy-hederagenin (specioside A) (Fig. S 1- S 8).
Compound 2 was obtained as a white amorphous powder with specific rotation of [α]D25: − 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 13C-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 acetylated hederagenin where compound 2 shows signal for C-23 at δC 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 1H and 13C-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 1H-NMR spectrum of compound 2 showed four anomeric proton signals at δH 4.20 (d, J = 8.0 Hz), 5.20 (brs), 5.21 (d, J = 6.4 Hz), 4.17 (d, J = 7.2), giving in the HSQC spectrum cross-peaks with four anomeric carbon signals at δC 104.8, 100.4, 94.5 and 103.4, respectively. HMBC correlations between δH 4.20 (d, J = 8.0 Hz) (H-1 of galactose) and δC 88.4 (C-3 of the aglycone), δH 5.20 (brs) (H-1 of rhamnose) and δC 74.6 (C-2 of galactose), δH 5.21 (d, J = 6.4 Hz) (H-1 of Glc I) and δC 175.7 (C-28 of aglycone), δH 4.17 (d, J = 7.2) (H-1 of Glc II) and δC 68.2 (C-6 of Glc I) showed the linkage points of the sugar molecules. The sugars in the structure were also proven by GC-MS analysis. Identification of L-rhamnose, D-galactose, and D-glucose was detected for 2, giving from peaks at 13.35, 15.89, and 17.09 min, respectively. Accordingly, the structure of 2 was assigned as 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl-oleanoic acid-28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (specioside B) (Fig. S 9- S 16).
Compound 3 was obtained as a white amorphous powder with a specific rotation of [α]D25 − 15.9 (c 0.13, MeOH). The IR and NMR data of the aglycon moiety of 3 were identical to the other new compounds (4). The C-3 oxymethine carbon and C-28 carbonyl carbon were observed at δC 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 1H-NMR spectrum of 3 displayed four anomeric proton signals at δH 4.26 (d, J = 7.8 Hz), 5.19 (brs), 5.18 (d, J = 8.4 Hz) and 4.17 (d, J = 7.8 Hz) giving in the HSQC spectrum cross-peaks with four anomeric carbon signals at δC 104.0, 100.3, 94.5 and 103.4, respectively. Proton signals for the sugar moieties were associated with COSY and HMBC spectra. Carbohydrate bonding points to aglycone were confirmed by HMBC correlations between δC 79.5 (C-3 of aglycone) and δH 4.26 (H-1 of xylose) and δC 175.7 (C-28 of aglycone) and δH 5.18 (H-1 of Glc I). The interactions between δC 76.6 (C-2 of xylose) and δH 5.19 (H-1 of rhamnose), δH 4.17 (H-1 of Glc II), and δC 68.1 (C-6 of Glc I) showed the bonding points of the sugar molecules to one another. Identification of L-rhamnose, D-xylose, and D-glucose in the GC-MS experiment was detected for 3, giving from peaks at 13.34, 14.58, and 17.09 min respectively, and thus the sugar types were clarified. Based on all this evidence IUPAC name of compound 3 is 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranosyl-hederagenin-28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (specioside C) (Fig.S 17- S 24).
Compound 4 was obtained as a pale yellow amorphous powder with a specific rotation of [α]D25 − 11.7 (c 0.34, MeOH). Compound 4 consists of 54 carbons according to 13C-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 δC 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 1H-NMR spectrum of compound 4 showed four anomeric proton signals at δH 4.26 (d, J = 7.2 Hz), 5.17 (brs), 5.20 (d, J = 8.4 Hz) and 4.18 (d, J = 8.0 Hz) giving HSQC spectrum cross-peaks with four anomeric carbon signals at δC103.8, 100.3, 94.5 and 103.4, respectively. HMBC cross-peaks between δC 80.0 (C-3 of aglycone) and δH 4.26 (H-1 of galactose), δC 175.7 (C-28 of aglycone) and δH 5.20 (H-1 of Glc I) showed the sugar linkage points to the aglycone. Furthermore, HMBC correlations between δH 5.17 (H-1 of rhamnose) and δC 74.4 (C-2 of galactose), δH 3.56, 3.90 (both H-6 of Glc I) and δC 103.4 (C-1 of Glc II) showed sugar to sugar bonding points. Identification of L-rhamnose, D-galactose, and D-glucose in GC-MS analysis was detected for 4, giving from peaks at 13.35, 15.89, and 17.09 min, respectively. Based on all this evidence IUPAC name of compound 4 is 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl-hederagenin-28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (specioside D) (Fig.S 25- S 41).
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 IC50 values was given in Table 3 and the percent vitality graph according to the MTT result was given in (Fig. S 42- S 49). According to the results, compound 2–4, and 3a did not have a cytotoxic effect on THP-1-derived macrophage cells. While, compound 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 IC50 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. (2021)28, 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 studies9,11. Furthermore, the structure of 1, 2a, and 4a compounds have the same galactose and rhamnose units attached third carbon of aglycone moieties however their aglycone structures are acetlylated hederagenin, olean type, and hederagenin, 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. Remarkably, 4a exhibit cytotoxic activity against A549, MDA-MB-231, PC-3, U-87 MG, and HeLa cell lines (8.13 ± 0.01, 8.43 ± 0.15, 18.81 ± 1.08, 63.19 ± 9.04, and 16.33 ± 4.75 µM) which has the highest significant effect when compared with others. These results may relate with free -CH2OH of hederagenin aglycone moiety of compound 4a increased the cytotoxicity. Moreover, acetlyted -OH units of hedereganin aglycone of compound 1 causes the notable cytotoxicity in comparison with oleanoic skeleton of compound 2.
Table 3
The IC50 values for compounds 1–4 and 2a-4a and doxorubicin (µM)
Samples | THP-1 originated-macrophage | CCD-34Lu | A549 | MDA-MB-231 | PC-3 | U87MG | HeLa | HepG-2 |
1 | 27.57 ± 0.52 | - | 8.59 ± 0.19 | 15.09 ± 1.02 | - | - | 48.11 ± 4.56 | - |
2 | - | - | - | - | - | - | - | - |
3 | - | - | - | - | - | - | - | - |
4 | - | - | - | - | - | - | - | - |
2a | 37.34 ± 8.17 | - | 20.77 ± 0.46 | 24.29 ± 2.65 | - | - | - | - |
3a | - | - | - | - | - | - | - | - |
4a | 35.74 ± 4.90 | - | 8.13 ± 0.01 | 8.43 ± 0.15 | 18.81 ± 1.08 | 63.19 ± 9.04 | 16.33 ± 4.75 | - |
Doxorubicin | 6.84 ± 0.18 | 33.98 ± 0.02 | 40.01 ± 0.02 | 70.00 ± 0.02 | 4.23 ± 0.02 | 11.09 ± 0.02 | 23.95 ± 0.02 | 36.85 ± 0.02 |
-: Not detected | | | | | | | |
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 29. Also, Genin et al.(2015)30 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 30000 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 and b). According to the fact that M1/M2 describes the two major and opposing activities of macrophages31. 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. Compounds 4a, 3a, and 1 showed the most distinguished MFI values with antigen treatment with 8706 ± 239.19, 9794.8 ± 291.84, and 9522.65 ± 283.68 values (***p < 0.001), respectively, among the other compounds when treated with IBV D274 antigen (Table 4). Moreover, Compounds 4a and 2a exhibited the highest CD163 MFI values without antigen treatment with MFI values of 8391.56 ± 264.75 and 7405.42 ± 224.13 (***p < 0.001), respectively (Table 5).
Table 4
MFI values of CD markers on THP-1 derived macrophage cells treated with saponins and antigen.a, b
|
CD 11b
|
CD80
|
CD163
|
THP-1 M0 + Ag
|
3,0274 ± 1,859.74
|
4,363.67 ± 133.29
|
6,827.7 ± 169.38
|
4 + Ag
|
32,925.3 ± 947.75
|
4,593.32 ± 951.6
|
8,203 ± 246.09 **
|
4a + Ag
|
30,124.7 ± 913.74
|
4,604.4 ± 128.49
|
8,706 ± 239.19 ***
|
2 + Ag
|
33,053.2 ± 951.6
|
4,516 ± 223.23
|
7,441 ± 423.23
|
2a + Ag
|
28,297.32 ± 808.92
|
4,727 ± 221.96
|
7,408.64 ± 224.16
|
3 + Ag
|
29,545.1 ± 906.36
|
4,564.51 ± 138.81
|
8,303.34 ± 245.1 **
|
3a + Ag
|
29,092.45 ± 852.78
|
4,911 ± 224.16
|
9,794.8 ± 291.84 ***
|
1 + Ag
|
31,644.21 ± 849.3
|
4,062.43 ± 906.36
|
9,522.65 ± 283.68 ***
|
a **P < 0.01; ***P < 0.001. |
b All values represented the mean ± standard deviation (n = 3test). |
Table 5
MFI values of CD markers on THP-1 derived macrophage cells treated with saponinsa,b
|
CD 11b
|
CD80
|
CD163
|
THP-1 M0
|
27,208 ± 2,444.77
|
4,476.31 ± 786.24
|
5,346 ± 205.83
|
4
|
27,324.7 ± 2,398.39
|
4,380 ± 169.38
|
6,755.64 ± 206.67**
|
4a
|
30,031.76 ± 1,486.58
|
4,552 ± 947.76
|
8,391.56 ± 264.75 **
|
2
|
29,875.39 ± 3,311.75
|
3,629.71 ± 142.8
|
6,206.45 ± 185.19
|
2a
|
26,304.81 ± 2,370.42
|
3,962.53 ± 246.09
|
7,405.42 ± 224.13 ***
|
3
|
28,092.69 ± 1,435.47
|
4,549.73 ± 913.74
|
6,655.82 ± 206.67 *
|
3a
|
29,092.75 ± 2,580.19
|
4,248 ± 137.13
|
6,408 ± 195.24 *
|
1
|
28,644.5 ± 849.33
|
4,536 ± 239.19
|
6,400.83 ± 196.02 *
|
a *P < 0.05; **P < 0.01; ***P < 0.001. |
b All values represented the mean ± standard deviation (n = 3test). |
Regarding saponins, studies suggest that they can trigger Th2 in M0 macrophages, which in turn stimulates M2 macrophage polarization32,33. 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. 201834 demonstrated that panax saponins promote M2 macrophage polarization so that depend on their effects of anti-inflammation, 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.