Analysis of the extracts from S. hexandrum before and after ultrafiltration with HPLC-UV/ESI-MS/MS analysis
Lignans and flavonoids are the two primary ingredients from S. hexandrum [3]. Prior to the ultrafiltration screening, the extracts from S. hexandrum were firstly subjected to HPLC-UV/ESI-MS/MS analysis in the positive ion mode, and each chromatographic peak was detected and tentatively identified in accordance with the retention time (Rt), UV spectra, protonated molecular fragment information ([M + H]+), characteristic fragment information, the corresponding standards and literature data. After the ultrafiltration screening of the extracts above with Topo I, Topo II, COX-2 and ACE2, the resulting samples were analyzed under the same LC-MS conditions. As a result, the chemical structures of 10 potential ligands binding to Topo I, Topo II, COX-2 and ACE2 were identified and summarized at great length in Table 1 and Fig. 1.
Table 1
Enrichment factors and LC-ESI-MS/MS data obtained from AUF screening of S. hexandrum with Topo I, Topo II, COX-2 and ACE2.
Compound
|
Rt (min)
|
EF (%)
|
[M + H]+
|
Characteristic fragment (m/z)
|
Identification
|
Topo I
|
Topo II
|
COX-2
|
ACE2
|
1
|
9.430
|
0.31
|
1.22
|
−
|
0.29
|
342
|
296, 279, 264, 236
|
Isocorydine b
|
2
|
13.329
|
1.85
|
0.68
|
0.20
|
0.07
|
611
|
611, 303
|
Rutin a
|
3
|
14.091
|
2.41
|
4.11
|
0.07
|
2.49
|
465
|
465, 303, 285
|
Quercetin 3-O-glucoside a
|
4
|
15.142
|
4.83
|
−
|
0.39
|
1.01
|
449
|
449, 287
|
Kaempferol 3-O-glucoside a
|
5
|
17.753
|
0.78
|
1.66
|
1.47
|
4.65
|
493
|
493, 397, 331, 317, 137
|
Unknown
|
6
|
18.588
|
3.98
|
−
|
0.70
|
0.47
|
397
|
397, 313, 282, 247, 229
|
β-Apopicropodophyllin b
|
7
|
19.737
|
1.24
|
13.90
|
0.33
|
−
|
303
|
303, 285, 229, 165, 153
|
Quercetin a
|
8
|
20.490
|
4.50
|
3.33
|
1.07
|
5.89
|
317
|
317, 302
|
Isorhamnetin a
|
9
|
22.297
|
0.51
|
7.06
|
1.58
|
0.43
|
287
|
287, 241, 213, 165, 153
|
Kaempferol a
|
10
|
22.911
|
7.03
|
−
|
1.35
|
1.49
|
415
|
397, 313, 282, 247, 229
|
Podophyllotoxin a
|
a Compared with the corresponding standard; b Identified based on the published literature. |
Among the potential bioactive components, the [M + H]+ ion at m/z 342 was considered as the molecular ion of compound 1, revealing the molecular formula of C20H23NO4. The MS/MS fragment ions comprised m/z 296 [M + H-CH3-OCH3]+, m/z 279 [M + H-(CH3)2NH-H2O]+, m/z 264 [M + H-(CH3)2-OCH3]+ and m/z 236 [M + H-(CH3)2-OCH3-CO]+. By comparing its MS/MS data with previous literature, compound 1 was recognized as isocorydine (341 Da) [29]. Compound 2 yielded the [M + H]+ ion at m/z 611 in the full scan mode. It produced characteristic fragment ions at m/z 303 that stemmed from the consecutive elimination of molecular 146 Da and 162 Da (rhamnosylglucoside). As regards compound 7, interestingly, the MS/MS spectra demonstrated the semblable result as compound 2, indicating the similar basic skeleton of flavonoids. Compound 7 exhibited the [M + H]+ ion at m/z 303. By comparing the Rt, the [M + H]+ ion and the MS/MS behaviors of corresponding reference standards, compounds 2 and 7 were successfully characterized as quercetin 3-rutinaside (rutin, calculated for C27H30O16, 610 Da) and quercetin (calculated for C15H10O7, 302 Da), respectively. And the MS/MS spectra of these two components were correspondent with the discussion in the known literature [30]. Concerning compound 3, the [M + H]+ ion was detected at m/z 465 in the full scan MS, and the molecular formula was deduced as C21H20O12. In addition, the neutral loss of a hexose moiety at m/z 162 generated the aglycon ion [M + H-Glu]+ at m/z 303. By comparison with the Rt and MS/MS information of the standard, compound 3 was thus identified as quercetin 3-O-glucoside (isoquercitrin, 464 Da). As for compound 4, the [M + H]+ ion was produced at m/z 449 and the molecular formula was regarded as C21H20O11. Typically, the aglycone ion at m/z 287 was formed by the neutral loss of a hexose moiety at m/z 287, tentatively inferred as kaempferol monoglycoside. By comparing the MS/MS data with the related reference standard, compound 4 was characterized as kaempferol 3-O-glucoside (astragalin, 448 Da), and the mass spectra were consistent with previously reported literature [26]. Compounds 6 and 10 presented the [M + H]+ ions at m/z 397 and m/z 415, and their molecular formulas were presumed to be C22H20O7 and C22H22O8, respectively. Furthermore, these two components possessed similar fragmentation pathways and MS/MS fragment information such as the ions at m/z 397, m/z 313 and m/z 282, derived from the loss of a water molecular moiety ([M + H-H2O]+), the characteristic retro Diels-Alder (RDA) cleavage ([M + H-H2O-C4H4O2]+) and the successive neutral loss of a methoxy moiety ([M + H-H2O-C4H4O2-OCH3]+), respectively. Moreover, the neutral losses of the C6H3(OCH3)3 moiety and a molecular of water moiety acquired the fragment ions at m/z 247 ([M + H-C6H3(OCH3)3]+) and m/z 229 ([M + H-C6H3(OCH3)3-H2O]+). Therefore, compounds 6 and 10 were further identified as β-apopicropodophyllin (396 Da) and podophyllotoxin (414 Da) in comparison to ESI-MS/MS spectra and characterized fragmentation pathways, combined with previous literature and the corresponding reference standards, respectively. Compounds 8 and 9 exhibited the [M + H]+ ions at m/z 317 and m/z 287, respectively. By comparing the ESI-MS/MS information of the existent reference standards and related literature reports [26], compounds 8 and 9 were unambiguously suggested as isorhamnetin (calculated for C16H12O7, 316 Da) and kaempferol (calculated for C15H10O6, 286 Da).
Screening of S. hexandrum for Topo I, Topo II, COX-2 and ACE2 ligands
Most medicinal plants have the characteristic of multiple drug targets for the treatment of various diseases. In order to further clarify the mechanism of action for medicinal plants, it is the first task to screen and identify active ingredients. Therefore, it is necessary to develop a fast, simple and effective approach for targeted screening of active components so as to associate the chemical ingredients and certain pharmacological activities. In addition, there are few reports on multi-targeted screening to date, which cannot meet the growing demand for multi-targeted screening of medicinal plant extracts. It is essential to accelerate the screening of multi-targeted medicinal plant activity for the discovery and development of new drugs. In this study, AUF-HPLC/MS with the characteristics of simplicity, efficiency and sensitivity was developed to screen Topo I, Topo II, COX-2 and ACE2 ligands from S. hexandrum, respectively. The variation in peak area for screened constituents could reflect a specific binding affinity between the activated and denatured enzymes before and after ultrafiltration. The enrichment factor (EF) represented the capacity for those ligands binding to target enzymes, and the calculation was shown as below:
EF (%) = (Aa-Ab) / A × 100%
where Aa, Ab and A represent peak areas of each chromatographic peak from the extract of S. hexandrum upon ultrafiltration with activated, denatured and without target enzymes (Topo I, Topo II, COX-2 and ACE2), respectively [31]. If the peak area from the activated group was greater than that of the corresponding inactivated group, those constituents were tentatively speculated as potentially bioactive ligands for target enzymes.
As illustrated in Figs. 2, 10, 7, 9 and 9 components from the extract of S. hexandrum exhibited specific bindings to Topo I, Topo II, COX-2 and ACE2 after AUF screening assay, which were deduced as potential ligands for Topo I, Topo II, COX-2 and ACE2, respectively. And the EF values of each component were shown in Table 1. The difference in the EF values of each compound indicated those potential ligands could specifically and differentially bind to the four target enzymes. That is, they possessed distinct and complicate interactions with the target enzymes. It also implied that different chemical components in the extracts could exert distinct but synchronous pharmaceutical effects in the pharmaceutical use as shown in Table 1.
In vitro anti-proliferative and COX-2 inhibitory assays of bioactive ligands screened
In order to explore the correlation between potential bioactive phytochemicals and pharmacological effects, anti-proliferative and COX-2 inhibitory assays in vitro were carried out so as to detect and validate their inhibitory effects of several bioactive ligands screened out targeting Topo and COX-2. For in vitro anti-proliferative assay, compounds 7, 9, 10 and 8 with relatively higher EF values exerted strong inhibitory activities (63.24%, 60.48%, 60.70%, and 32.76%, respectively) on A549 cell at the concentration of 100 µM, and were comparable with the positive controls of etoposide and 5-FU (71.13% and 50.44%, respectively). Moreover, compounds 10 and 7 also showed remarkable inhibitory effects (46.36% and 40.47%, respectively) on HT-29 cell at the concentration of 100 µM, whereas etoposide and 5-FU were 32.24% and 10.95%, respectively. With regard to in vitro COX-2 inhibitory assays, compared with the positive control of indomethacin at 0.73 ± 0.07 µM, compounds 9 and 10 with higher EF values showed significant inhibitory effects with IC50 value at 0.36 ± 0.02 µM and 10.49 ± 0.61 µM, respectively. Hence, it was well worth fishing out and identifying potential bioactive ligands from S. hexandrum combining its empirical applications.
Molecular docking simulation
Molecular docking studies were implemented to further simulate the target proteins and several representative compounds with the highest EF values. We applied the AutoDockTools 1.5.6 and Discovery Studio 4.5 Client software with the three-dimensional crystal structures of Topo I, Topo II, COX-2 and ACE2 in this work, respectively. During this simulation procedure, the grid box dimensions and the centroid coordinate for molecular docking of the macromolecular target proteins were shown in Table 2. The molecular docking results of several representative compounds and positive drugs against Topo I, Topo II, COX-2 or ACE2 were also displayed in Fig. 4 and Table 3.
Table 2
Dimensions and centroid coordinates of grid box the macromolecular target proteins in the molecular docking analysis.
Protein targets
|
PDB ID
|
Dimension of the grid box
(npts)
|
Center grid box
(xyz coordinates)
|
Grid box spacing
(angstrom)
|
Topo I
|
1T8I
|
60 ⋅ 60 ⋅ 60
|
21.474, -2.226, 27.863
|
0.375
|
Topo II
|
3QX3
|
60 ⋅ 60 ⋅ 60
|
33.026, 95.765, 51.567
|
0.375
|
COX-2
|
1CX2
|
60 ⋅ 60 ⋅ 60
|
24.263, 21.528, 16.497
|
0.375
|
ACE2
|
1R42
|
60 ⋅ 60 ⋅ 60
|
52.874, 68.399, 33.501
|
0.375
|
Table 3
The molecular docking results of several representative compounds screened from S. hexandrum and positive drugs against Topo I, Topo II, COX-2 or ACE2.
No.
|
Phytochemicals
|
Drug targets
|
BE (kcal/mol)
|
IC50 (µM)
|
H-bond atoms
|
1
|
Podophyllotoxin
|
Topo I
|
-6.32
|
23.25
|
Dc112, Lys425 and Met428
|
2
|
Quercetin
|
Topo II
|
-6.99
|
7.53
|
Dg13, Dt9, Gln778 and Asp479
|
3
|
Kaempferol
|
COX-2
|
-7.22
|
5.10
|
Tyr355, Gln192 and Gly526
|
4
|
Isorhamnetin
|
ACE2
|
-5.72
|
63.95
|
Glu375 and Ala348
|
5
|
Camptothecin a
|
Topo I
|
-7.57
|
2.85
|
Da113, Dc112, Glu356, Lys425
|
6
|
5-FU b
|
Topo I
|
-3.70
|
1950.00
|
Met428, Tyr426
|
7
|
Etoposide c
|
Topo II
|
-7.62
|
2.59
|
Da12, Gln778
|
8
|
Indomethacin d
|
COX-2
|
-9.18
|
0.19
|
Glu524
|
9
|
MLN-4760 e
|
ACE2
|
-4.27
|
738.62
|
Glu375, Glu402, Thr371
|
a, b, c, d, e positive control. |
As shown in Table 3, podophyllotoxin exhibited a higher affinity to Topo I as its binding energy (BE) and the theoretical IC50 values were − 6.32 kcal/mol and 23.25 µM, lower than the positive control 5-FU (-3.70 kcal/mol and 1.95 mM) and slightly higher than Topo I inhibitor camptothecin (-7.57 kcal/mol and 2.85 µM). Considering Topo II, quercetin with a larger EF value displayed a strong affinity to Topo II with the BE of -6.99 kcal/mol and the theoretical IC50 value of 7.53 µM, which was comparable to Topo II inhibitor etoposide (-7.62 kcal/mol and 2.59 µM). In addition, kaempferol exhibited a higher affinity to COX-2, and its BE and the theoretical IC50 values were calculated as -7.22 kcal/mol and 5.10 µM, which was not much different from the positive control indomethacin (-9.18 kcal/mol and 186.44 nM). With regard to ACE2, isorhamnetin was discovered with a high binding affinity of -5.72 kcal/mol and the theoretical IC50 value of 63.95 µM, lower than that of ACE2 inhibitor MLN-4760 (-4.27 kcal/mol and 738.62 µM). Above all, molecular docking analysis indicated that some bioactive ligands screened with the largest EF values were provided with relatively lower binding energies and inhibitory effects compared with the positive controls or the same group combining other bioactivity assays in vitro such as anti-proliferative and COX-2 inhibitory assays. The docking results were consistent with the ultrafiltration screening and in vitro bioactivity verification results, further confirming the feasibility of the molecular docking approach.