2.1. Anti-trypanosomal activity
Using the protocol of Huber and Koella, the total ethanol extract and its derived fractions; petroleum ether, and ethyl acetate of Nephthea mollis were tested for their in-vitro anti-trypanosomal results against T. brucei revealed that the total ethanol extract and ethyl acetate fraction of the soft coral Nephthea mollis displayed remarkable in-vitro anti-trypanosomal potential against Trypanosoma brucei with IC50 value of 6.4 and 3.7 (µg/ml, 72 h) respectively. Unfortunately, the Petroleum ether fraction shows a weak anti-trypanosomal activity with IC50 value of > 50 (µg/ml, 72 h).
2.2. Metabolomic analysis
The total ethanol extract of Nephthea mollis was subjected to metabolomic profiling, for the first time and the chemical profiling revealed various classes of secondary metabolites, where sesquiterpenes were the most abundant, in addition to diterpenes and terpenoid-related carboxylic acids (Figs.1 and 2). The detected compounds were identified by using macros, algorithms using MZmine, and online databases (DNP, METLIN, and Marinlit databases) [17]. From these databases, thirty-three compounds were identified (Table 1; Fig.3). Fig.1. Total ion chromatogram of total extract of Nephthea mollis (Negative mood).
The mass ion peak at m/z 204.187 for the suggested formula C15H24 was identified as Cadina-4(14),5-diene (1) which was formerly reported from the soft coral Nephthea sp. [18]. Likewise, the mass ion peak at m/z 204.187 for the predicted molecular formula C15H24 was also dereplicated as the sesquiterpene; 4,15-dihydro guaian-6,10-diene (2), which was previously obtained from Nephthea chabrolii [19]. Additionally, the mass ion peak at m/z 220.183 in accordance with the molecular formula C15H24O, was dereplicated as eudesma-4,7(11)-diene-8β-ol (3) and/or guaianediol (4) and/or 1S,4R,5S-guia-6,9-dien-4-ol (5) and/or 3,4-epoxyguaia-10(12)-ene (6) and/or guaian-4,6-dien-10α-ol (7) and/or 8β-hydroxyprespatane (8) and/or capillosanol (9), all of these sesquiterpenes were previously obtained from Nephthea chabrolii [20] [21] [19], except sesquiterpenes (6) and (8) were obtained from Nephthea sp. and Nephthea erecta respectively [22] [23]. Similarly, the mass ion peak at m/z 234.161 for the predicted molecular formula C15H22O2, was dereplicated as the sesquiterpenes; 1α-hydroxy-(+)-cyclocolorenone (10), which was formerly characterized from Nephthea sp. [24]. Furthermore, the mass ion peak at m/z 236.177 in accordance with the molecular formula C15H24O2, was dereplicated as hydroxycolorenone (11) and/or 6β,7β-epoxy-4β-hydroxyguaian-10-ene (12) and/or 3,4-epoxy-11-hydroxy-1-pseudoguaiene (13) and/or 8β–hydroperoxyprespatane (14) and/or chabrolidione A (15) and/or (2E,6E)-3-isopropyl-6-methyl-10-oxoundec-2,6-dienal (16), sesquiterpenes (11), (12) and (15) were previously obtained from Nephthea chabrolii [24] [19] [25], while sesquiterpenes (13), (14) and (16) were obtained from Nephthea erecta [19] [22] [26]. Moreover, the mass ion peak at m/z 238.193 in agreement with the molecular formula C15H26O2 was dereplicated as the sesquiterpenes; ent-oplopanone (17) and/or nephthediol (18), both of them were previously reported from Nephthea sp. [18]. Similarly, the mass ion peak at m/z 240.172 for the predicted molecular formula C14H24O3, was dereplicated as the sesquiterpenes; chabranol (19), which was formerly characterized from Nephthea chabrolii [23]. Moreover, the mass ion peak at m/z 250.193 in agreement with the molecular formula C16H26O2 was dereplicated as the sesquiterpenes; Methoxycolorenone (20) and/or 2-deoxy-7-O-methyllemnacarnol (21), the former was previously reported from Nephthea chabrolii [27], while the other was previously reported from Nephthea sp. [28]. Another compound was dereplicated as 10α-methoxy-4β-hydroxy guaian-6-ene (22), on account of the observed mass ion peak at m/z 252.208, and in accordance with the molecular formula C16H28O2, this sesquiterpene was formerly characterized from Nephthea chabrolii [19].
Table 1
A list of identified metabolites in soft coral Nephthea mollis.
|
Compound
|
Class
|
Rt
|
Molecular
Formula
|
Mass
|
1
|
Cadina-4(14),5-diene
|
Sesquiterpenes
|
6.12
|
C15H24
|
204.1875
|
2
|
4,15-dihydro guaian-6,10-diene
|
Sesquiterpenes
|
6.12
|
C15H24
|
204.1879
|
3
|
Eudesma-4,7(11)-diene-8β-ol
|
Sesquiterpenes
|
4.73
|
C15H24O
|
220.183
|
4
|
Guaianediol
|
Sesquiterpenes
|
4.73
|
C15H24O
|
220.183
|
5
|
1S,4R,5S-guia-6,9-dien-4-ol
|
Sesquiterpenes
|
4.73
|
C15H24O
|
220.183
|
6
|
3,4-epoxyguaia-10(12)-ene
|
Sesquiterpenes
|
4.73
|
C15H24O
|
220.183
|
7
|
guaian-4,6-dien-10α-ol
|
Sesquiterpenes
|
4.73
|
C15H24O
|
220.183
|
8
|
8β-hydroxyprespatane
|
Sesquiterpenes
|
4.73
|
C15H24O
|
220.183
|
9
|
Capillosanol
|
Sesquiterpenes
|
4.73
|
C15H24O
|
220.183
|
10
|
1α-hydroxy-(+)-cyclocolorenone
|
Sesquiterpenes
|
2.99
|
C15H22O2
|
234.1614
|
11
|
Hydroxycolorenone
|
Sesquiterpenes
|
2.99
|
C15H24O2
|
236.1774
|
12
|
6β,7β-Epoxy-4β-hydroxyguaian-10-ene
|
Sesquiterpenes
|
2.99
|
C15H24O2
|
236.1774
|
13
|
3,4-epoxy-11-hydroxy-1-pseudoguaiene
|
Sesquiterpenes
|
2.99
|
C15H24O2
|
236.1774
|
14
|
8β-hydroperoxyprespatane
|
Sesquiterpenes
|
2.99
|
C15H24O2
|
236.1774
|
15
|
Chabrolidione A
|
Sesquiterpenes
|
2.99
|
C15H24O2
|
236.1774
|
16
|
(2E,6E)-3-isopropyl-6-methyl-10-oxoundec -2,6-dienal
|
Sesquiterpenes
|
2.99
|
C15H24O2
|
236.1774
|
17
|
Ent-oplopanone
|
Sesquiterpenes
|
4.72
|
C15H26O2
|
238.1934
|
18
|
Nephthediol
|
Sesquiterpenes
|
4.72
|
C15H26O2
|
238.1934
|
19
|
Chabranol
|
Sesquiterpenes
|
4.50
|
C14H24O3
|
240.1727
|
20
|
Methoxycolorenone
|
Sesquiterpenes
|
5.21
|
C16H26O2
|
250.1932
|
21
|
2-deoxy-7-O-methyllemnacarnol
|
Sesquiterpenes
|
5.21
|
C16H26O2
|
250.1932
|
22
|
10α-methoxy-4β-hydroxy guaian-6-ene
|
Sesquiterpenes
|
5.01
|
C16H28O2
|
252.2085
|
23
|
Nephtheoxydiol
|
Sesquiterpenes
|
5.11
|
C15H26O3
|
254.1878
|
24
|
1S-acetoxygermacra-3Z,5E,10(15)-triene
|
Sesquiterpenes
|
5.41
|
C17H26O2
|
262.1932
|
25
|
Armatin E
|
Sesquiterpenes
|
5.41
|
C16H24O3
|
264.1718
|
26
|
4α-O-acetyl-selin-11-en
|
Sesquiterpenes
|
5.41
|
C17H28O2
|
264.2088
|
27
|
2-deoxy-12-ethoxy-7-O-methyl lemnacarnol
|
Sesquiterpenes
|
5.99
|
C18H30O3
|
294.2187
|
28
|
Ketochabrolic acid
|
Terpenoid-related carboxylic acids
|
2.95
|
C18H28O3
|
292.2032
|
29
|
Isoketochabrolic acid
|
Terpenoid-related carboxylic acids
|
2.95
|
C18H28O3
|
292.2032
|
30
|
Chabrolol A
|
Diterpenes
|
5.41
|
C17H28O2
|
264.2088
|
31
|
Brassicolide
|
Diterpenes
|
5.65
|
C20H30O3
|
318.2202
|
32
|
Brassicolene
|
Diterpenes
|
5.73
|
C22H32O2
|
328.2408
|
33
|
Nephthenol
|
Diterpenes
|
2.99
|
C20H34O
|
290.260
|
2.3. Molecular docking
For further exploration and getting a better idea about the possible target affected by dereplicated compounds of Nephthea mollis to afford their anti-trypanosomal activity, we performed in-silico molecular docking [2] simulations within ornithine decarboxylase (ORD). The X-ray structure of ORD showed to ligands; D-Ornithine, ORX, a substrate analog and was placed within ORD active site and Geneticin (G418) as a weak non-competitive inhibitor occupying allosteric site of ORD. Molecular docking simulations performed within the active site of ORD should number of binding interactions (H-bonding and H-pi interactions) between ORX and a number of amino acid residues (GLU 274, ASP 332, GLY 276, ARG 277, TYR 389, GLY 237, and HIS 197) with binding free energy (S) of -4.5058 kcal/mol, (Fig. 5). On the other hand, five of the identified compounds (2-deoxy-12-ethoxy-7-O-methyl lemnacarnol, Nephthenol, 4α-O-acetyl-selin-11-en, Eudesma-4,7(11)-diene-8β-ol, and Chabrolidione A) showed a better binding interaction with binding free energy lower than that found with the substrate analog, ORX, (Table 2).
Table 2
Binding free energy (S; kcal/mol) and binding accuracy (RMSD; Å) of Nephthea mollis and co-crystallized ligand within ornithine decarboxylase (ORD) active site (PDB ID: 1NJJ; 2.45 Å)
Molecule
|
Energy score
(S; kcal/mol)
|
RMSD (Å)
|
INJJ Co-crystallized ligand (ORX)
|
- 4.5058
|
1.8918
|
2-deoxy-12-ethoxy-7-O-methyl lemnacarnol
|
-5.2186
|
2.0242
|
Nephthenol
|
-4.8930
|
1.8370
|
4α-O-acetyl-selin-11-en
|
-4.6440
|
1.9708
|
Eudesma-4,7(11)-diene-8β-ol
|
-4.5653
|
0.6331
|
Chabrolidione A
|
-4.5278
|
0.9897
|
Guaian-4,6-dien-10α-ol
|
-4.4267
|
1.6648
|
Brassicolide
|
-4.4262
|
1.8294
|
Capillosanol
|
-4.2383
|
2.2662
|
Chabrolol A
|
-4.2271
|
1.8498
|
Nephtheoxydiol
|
-4.1673
|
1.4596
|
1α-hydroxy-(+)-cyclocolorenone
|
-4.0963
|
1.3961
|
1S-acetoxygermacra-3Z,5E,10(15)-triene
|
-4.0880
|
0.9902
|
8β-hydroperoxyprespatane
|
-4.0673
|
1.5743
|
3,4-epoxyguaia-10(12)-ene
|
-4.0394
|
1.7450
|
2-deoxy-7-O-methyllemnacarnol
|
-4.0057
|
1.4953
|
4,15-dihydroguaian-6,10-diene
|
-3.9922
|
1.9155
|
10α-methoxy-4β-hydroxy guaian-6-ene
|
-3.9431
|
1.8772
|
8β-hydroxyprespatane
|
-3.8998
|
1.5906
|
6β,7β-Epoxy-4β-hydroxyguaian-10-ene
|
-3.8558
|
2.0234
|
Methoxycolorenone
|
-3.7555
|
2.1270
|
Chabranol
|
-3.7507
|
1.6197
|
Hydroxycolorenone
|
-3.5285
|
2.1016
|
Nephthediol
|
-3.4163
|
2.4168
|
Cadina-4(14),5-diene
|
-3.4067
|
2.4022
|
Armatin E
|
-3.3336
|
2.1020
|
Ent-oplopanone
|
-2.8246
|
2.3728
|
Brassicolene
|
-2.7567
|
2.5090
|
1S,4R,5S-guia-6,9-dien-4-ol
|
-2.0255
|
1.1860
|
Moreover, most of the remaining compounds were almost equal to ORX binding free energy or a little bit lower, as listed in Table 2. 6,7-epoxy-4-hydroxyguaian-10-ene, chabrolidione A, and nephtheoxydiol showed a good overlay with co-crystallized ligand ORX within ORD active site (Fig. 4).
Furtherly, visual inspection of the resultant docking poses of each compound showed a number of binding interactions (varying from H-bonding to H-pi interactions) between some of the dereplicated compounds and various amino acids lining ORD active site similar to that found with ORX, (Table 3; Fig. 5). All in all, the obtained molecular docking data showed how good the overlay of the dereplicated compounds of Nephthea mollis within ornithine decarboxylase (ORD) active site, which could explain their anti-trypanosomal activity against Trypanosoma brucei.
Table 3
Binding free energy (S; kcal/mol) and binding interactions for co-crystallized ligand (ORX) and Nephthea mollis within ornithine decarboxylase (ORD) active site (PDB ID: 1NJJ; 2.45 Å)
Ligand
|
Binding
energy score
(S; kcal/mol)
|
Ligand – active site interactions
|
a. a. residue
|
Bond type
|
Bond length (Å)
|
Co-crystallized
Ligand (ORX)
|
- 4.5058
|
GLU 274
|
H-donor
|
3.16
|
ASP 332
|
H-donor
|
3.30
|
GLY 276
|
H-acceptor
|
2.92
|
ARG 277
|
H-acceptor
|
2.74
|
TYR 389
|
H-acceptor
|
2.55
|
GLY 237
|
H-acceptor
|
2.70
|
GLY 276
|
H-acceptor
|
3.07
|
TYR 389
|
H-pi
|
3.71
|
HIS 197
|
Pi-pi
|
3.88
|
8β-hydroxyprespatane
|
-3.8998
|
ARG 154
|
H-acceptor
|
2.91
|
ARG 154
|
H-acceptor
|
3.17
|
Capillosanol
|
-4.2383
|
HIS 197
|
H-pi
|
4.36
|
1α-hydroxy-(+)-cyclocolorenone
|
-4.0963
|
HIS 197
|
H-pi
|
3.98
|
Hydroxycolorenone
|
-3.5285
|
LYS 69
|
H-acceptor
|
3.11
|
6β,7β-Epoxy-4β-hydroxyguaian-10-ene
|
-3.8558
|
LYS 69
|
H-acceptor
|
2.95
|
HIS 197
|
H-pi
|
3.97
|
8β-hydroperoxyprespatane
|
-4.0673
|
LYS 69
|
H-acceptor
|
3.31
|
Chabranol
|
-3.7507
|
ARG 277
|
H-acceptor
|
2.91
|
HIS 197
|
H-pi
|
3.85
|
Chabrolidione A
|
-4.5278
|
LYS 69
|
H-acceptor
|
2.94
|
ARG 277
|
H-acceptor
|
2.92
|
SER 200
|
H-acceptor
|
3.06
|
1S-acetoxygermacra-3Z,5E,10(15)-triene
|
-4.0880
|
GLU 274
|
H-donor
|
3.31
|
Chabrolol A
|
-4.2271
|
GLU 274
|
H-donor
|
2.71
|
Nephthenol
|
-4.8930
|
ARG 277
|
H-acceptor
|
2.79
|
Nephthediol
|
-3.4163
|
LYS 69
|
H-acceptor
|
3.26
|