2.2. Chemical Composition
The HPLC-DAD and LC-MS (ESI) analysis of the DvA
n-hexane soluble fraction allowed us to indentify five compounds (
Figure 1 and
Figure 2;
Table 2).
Figure 1 shows the HPLC profiles at 206 and 254 nm of the DvA extract under the optimized conditions of analysis, whereas
Figure 2 shows the LC-MS (ESI) profile in positive and negative modes.
Table 2 lists the compounds identified with their UV absorption maxima, molecular weights and characteristic adducts. The chromatograms in
Figure 1 at 206 nm show the presence of two main peaks tomentosin (2) and costic acid (4). Furthermore, three minor peak compounds
1,
3,
5 were found. These peaks could be identified as inuviscolide, ilicic acid and 3α-hydroxycostic acid, respectively.
Peak 1, identified as inuviscolide, presents a λ
max at 225 and 254 nm and a positive and negative ESI-MS spectrum comparable with compound
2, tomentosin. Tomentosin presents a λ
max at 254 nm, attributable to its flavonoid chromophore. The positive ESI-MS spectrum of tomentosin exhibits the signals at
m/z 231 [M-H
2O+H]
+,
m/z 249 [M+H]
+ m/z and 271 [M+Na]
+ and negative ESI-MS ion at
m/z 247 [M-H]
- (
Figure 2). Inuviscolide and tomentosin are sesquiterpene lactones with cytotoxic and antibacterial activities isolated from
D. graveolens [
41]. Peak
3, identified as ilicic acid (eudesmane sesquiterpene derivative) showed a λ
max at 227 nm and gives the positive mass signals at
m/z 275 [M+Na]
+ and
m/z 235 [M-H
2O+H]
+ and the negative signal at
m/z 251 [M-H]
- (
Figure 2).
Figure 1.
UV spectra at 206 and 254 nm of DvA with UV spectra of 1) inuviscolide, 2) tomentosin, 3) ilicic acid, 4) costic acid, 5) 3α-hydroxy costic acid.
Figure 1.
UV spectra at 206 and 254 nm of DvA with UV spectra of 1) inuviscolide, 2) tomentosin, 3) ilicic acid, 4) costic acid, 5) 3α-hydroxy costic acid.
Figure 2.
ESI total ion chromatogram of of DvA extract and selected ion monitoring for each compound.
Figure 2.
ESI total ion chromatogram of of DvA extract and selected ion monitoring for each compound.
Table 2.
Main signals exhibited in the HPLC-MS2 spectra of compounds detected in D. viscosa extract and UV Maxima (λ max) detected in HPLC-DAD analysis and proposed attributions.
Table 2.
Main signals exhibited in the HPLC-MS2 spectra of compounds detected in D. viscosa extract and UV Maxima (λ max) detected in HPLC-DAD analysis and proposed attributions.
Compound | Number | λ max | mol. wt. | LC-MS (ESI)
m/z (amu) |
---|
inuviscolide | 1 | 225, 254 | 248 | 231 [M-H2O+H]+; 249 [M+H]+; 271 [M +Na]+ |
tomentosin | 2 | 254 | 248 | 231 [M-H2O+H]+; 249 [M+H]+; 271 [M +Na]+; 247 [M-H]- |
ilicic acid | 3 | 227 | 252 | 275 [M+Na]+; 235 [M-H2O+H]+ ; 251 [M-H]- |
costic acid | 4 | 210 | 234 | 235 [M+H]+ |
3α-hydroxycostic acid | 5 | 210 | 251 | 274 [M+Na]+ |
For the high resolution mass spectrometry analysis of pure standards, tomentosin and inuviscolide showed similar spectra with [M+H]
+, [M+H-H
2O]
+ and [M+NH
4]
+ adducts. The ppm difference between an observed ion mass/charge and exact ion mass/charge was below 10.03 (
Figure 5,
Table 3).
Ilicic acid had been reported as one of the active anti-inflammatory principles of
D. viscosa [
42,
43]. Costic acid (peak
4) presents a λ
max at 210 nm in ESI
+ mode at
m/z 257 [M+Na]
+ while its hydroxyl-derivative 3α hydroxycostic acid (peak
5) gives a signal at
m/z 252 [M+H]
+ (
Figure 2).
D. viscosa has been repeatedly studied from the chemical point of view and several sesquiterpene lactones, sesquiterpene acids and flavonoids have been isolated from specimens of diverse origins [
14,
15,
16,
17,
18,
19].
Table 3.
Q-TOF (ESI+) characteristic of inuviscolide, tomentosin, ferunelol. *calculated by Chemdraw Pro 8.0, Cambridge Soft.
Table 3.
Q-TOF (ESI+) characteristic of inuviscolide, tomentosin, ferunelol. *calculated by Chemdraw Pro 8.0, Cambridge Soft.
Compound | Molecular formula | r. t. (min.) | Log P* | Calculated mass (amu) [M+H]+ | Measured mass (amu) [M+H]+ | Error (ppm) |
---|
inuviscolide | C15H20O3 | 4.628 | 2.15 | 249.1485 | 249.1454 | 10.03418 |
tomentosin | C15H20O3 | 4.775 | 1.56 | 249.1485 | 249.1477 | 3.210936 |
ferulenol | C24H30O3 | 10.020 | 5.68 | 367.2268 | 367.2263 | 1.361556 |
Figure 3.
Spectra at 257 of FcA and FcR with UV spectra of 6) acetoxyferutinin, 7) unknown, 8) oxojaeskeanadioyl anisate, 9) fertidin and 10) ferulenol.
Figure 3.
Spectra at 257 of FcA and FcR with UV spectra of 6) acetoxyferutinin, 7) unknown, 8) oxojaeskeanadioyl anisate, 9) fertidin and 10) ferulenol.
The chromatographic analysis of FcA and FcR
n-hexane dissolved fraction allowed to identify three daucanes and one coumarin compound (
Figure 3 and
Figure 4;
Table 4).
Figure 3 shows the HPLC-UV profile at 257 nm and
Figure 4 illustrates the LC-MS (ESI) profile in positive mode of both FcA and FcR extracts. The chromatogram shows the presence of one main peak 7 and four other peaks 6, 8, 9 and 10 (
Figure 3). Peaks 6, 8, 9 and 10 could be identified as acetoxy ferutinin, oxo-jaeskeanadiol anisate, fertidin and ferulenol, respectively. The UV spectrum of 7 presents λ
max at 208 nm while in positive and negative ESI-MS spectra no spectra were recorded because of that we could not be certain that is lapiferin. Peaks 6, 8 and 9 show UV spectrum of λ
max at 257 nm. The ESI negative spectra of compound 6 showed a signal at
m/z 415 [M-H]
-, and the ESI positive spectra of 8 and 9 compounds showed the
m/z 409 [M+Na]
+ and
m/z 439 [M-H
2O+H]
+, respectively. Compounds 6, 8 and 9 were, therefore, identified as acetoxy ferutinin, oxo-jaeskeanadioil anisate and fertidin, respectively (
Table 4). The UV spectrum of 10 shows λ
max 202, 282 and 307 nm and the positive ESI spectra showed [M+H]
+ signal at
m/z 367 and [M+Na]
+ signal at
m/z 389 while the [M-H]
- ion at
m/z 365 was recorded in the negative ESI-MSspectrum (
Figure 4) and was identified as ferulenol. The high resolution mass spectrometry analysis of pure standard showed only [M+H]
+ and [M+NH
4]
+ adducts (
Figure 5). The ppm difference between an observed ion mass/charge and exact ion mass/charge was 1.36 (
Table 3).
In the FcA extract it is evident lower signals intensity attributed to identified compound in the respect to FcR extract (
Figure 4). The differences in the chemical composition between leaves, steam, roots and latex have been reported previously [
15]. The components of FcA and FcR extracts were identified by comparing their HPLC retention time, UV absorption maxima and positive and negative mode fragmentation patterns to the corresponding data reported by Arnoldi
et al. [
14].
Figure 4.
ESI total ion chromatogram of Ferula communis extracts and selected ion monitoring for each compound.
Figure 4.
ESI total ion chromatogram of Ferula communis extracts and selected ion monitoring for each compound.
Table 4.
Main signals exhibited in the HPLC-MS2 spectra of compounds detected in F. communis extracts and UV Maxima (λ max) detected in HPLC-DAD analysis and the proposed attributions.
Table 4.
Main signals exhibited in the HPLC-MS2 spectra of compounds detected in F. communis extracts and UV Maxima (λ max) detected in HPLC-DAD analysis and the proposed attributions.
Compound | Number | λ max | mol. wt. | LC-MS (ESI)
m/z (amu) |
---|
acetoxy ferutinin | 6 | 257 | 416 | 415 [M-H]- |
oxojaeskeanadioyl anisate | 8 | 257 | 386 | 409 [M+Na]+ |
fertidin | 9 | 257 | 456 | 439 [M-H2O+H]+ |
ferulenol | 10 | 202, 282, 307 | 366 | 367 [M+H]+; 365 [M-H]-; 389 [M+Na]+ |
Figure 5.
LC-Q-TOF spectra of A) tomentosin, B) inuviscolide, C) ferulenol.
Figure 5.
LC-Q-TOF spectra of A) tomentosin, B) inuviscolide, C) ferulenol.
The quantity of inuviscolide and tomentosin from
D. viscosa and ferulenol from
F. communis extracts expressed as mg/g of extracts are presented in
Table 5.
Table 5.
Content of inuviscolide, tomentosin and ferulenol in plant extracts.
Table 5.
Content of inuviscolide, tomentosin and ferulenol in plant extracts.
Extract | Compounds |
---|
inuviscolide | tomentosin | ferulenol | others |
---|
(mg/g extract) | (%) |
---|
DvA | 42.68 | 205.80 | - | 75.15 |
FcR | - | - | 88.40 | 91.16 |
FcA | - | - | 28.20 | 97.18 |
2.3. Antifungal Activities
Data of ED
50s of the extracts on colony growth and conidial germination inhibitions are presented in
Table 6.
Table 6.
ED50 (mg/L) and MIC (mg/L, between the brackets) of the extracts on colony growth inhibitions after two (B. fuckeliana), six (P.digitatum), seven (M. laxa and M. fructigena), nine (Aspergillus spp. and P. expansum) days and conidial germination inhibitions after 10-12 h of incubations. M: colony growth; C: conidial germination; nd: not determined due to lack of inhibition; ─ not tested.
Table 6.
ED50 (mg/L) and MIC (mg/L, between the brackets) of the extracts on colony growth inhibitions after two (B. fuckeliana), six (P.digitatum), seven (M. laxa and M. fructigena), nine (Aspergillus spp. and P. expansum) days and conidial germination inhibitions after 10-12 h of incubations. M: colony growth; C: conidial germination; nd: not determined due to lack of inhibition; ─ not tested.
Extracts | Aspergillius spp. | B. fuckeliana | P. expansum | P. digitatum | M. laxa | M. fructigena |
---|
M | C | M | C | M | C | M | C | M | C | M | C |
---|
DvA | >400 | 371 | 153 | 192 | >400 | 201 | 154 | 83 | 25 | 106 | 29 | 94 |
| (nd) | (400) | (400) | | (400) | (400) | (400) | (200) | (300) | (200) | (300) |
FcR | >400 | nd | 136 | >400 | >400 | nd | >400 | >400 | 202 | nd | 75 | nd |
| | (400) | | | | | | (400) | | (400) | |
FcA | >400 | ─ | >400 | ─ | >400 | ─ | >400 | ─ | >400 | ─ | >400 | ─ |
DvA was found to inhibit the colony growth of the fungi in a dose dependent manner. The highest antifungal activity of this extract was found on
M. laxa and
M. fructigena with ED
50 25.4 and 29 mg/L respectively, followed by
B. fuckeliana (ED
50 153 mg/L) and
P. digitatum (ED
50 154 mg/L). The lowest antifungal activity of this extract was found on
Aspergillus spp. and on
P. expansum andcalculated ED
50s were higher than the range of the used concentrations. DvA extract showed inhibition of conidial germination of all tested fungi. The ED
50s of 371, 192, 201, 83, 106 and 94mg/L were calculated for
Aspergillus spp,
B. fuckeliana,
P. expansum, P. digitatum, M. laxa and
M. fructigena, respectively. DvA demonstrated relatively high activity on conidia germination but very low activity on colony growth inhibition of
Aspergillus spp. and
P. expansum. These results confirm the results found by Abou-Jawdah
et al. [
35] where extracts of
D. viscosa showed high activity against spore germination but only moderate activity against mycelial growth. There are different publications that demonstrate the activity of
D. viscosa extracts against dermatophytes [
30,
31,
32,
33] and fungal pathogens [
35,
45,
46] under
in vitro conditions. However, there are no data that reports activities of
D. viscosa extract on
M. laxa and
M. fructigena, even though in our experimental conditions it showed the highest effects. FcR show very low activity on the colonies of
Aspergillus spp.,
P. expansum and
P. digitatum whereas, oncolony growthof
M. fructigena, B. fuckeliana and
M. laxa it was found to have a good fungitoxic effect with ED
50 of 75, 135 and 202 mg/L, respectively. No inhibitory activity of FcR was found on conidial germination in all tested fungi. FcA extract showed neglected inhibitory activity on tested fungi regarding colony growth inhibition under our experimental conditions. Therefore, the activity of this extract was not tested against the conidial germination. MICs values in most cases were of 400 mg/L except for
M. laxa and
M.fructigena of 200 and 300 mg/L for mycelia and conidia, respectively. In the case of
Aspergillus spp. at 400 mg/L only 90% of mycelia growth inhibition was archived and MIC was not determined.
Principal component analysis (PCA) on the colony growth inhibitions and cluster analysis separate extracts into four different groups (
Figure 6).
Figure 6 shows that the principal components PC1 and PC2 accounted for 98.51% of the variation. Based on PCA and cluster analysis, I group contains DvA 3,4, FcR extracts, II DvA 2, 6, FcR 2,3, III DvA 1, FcA 2,4, FcR 1,6 and IV DvA 5, FcA 3, 5, 6, FcR 5 (
Figure 6).
Figure 6.
Classification of plant extracts according to colony growth of six postharvest fungi by PCA and cluster analysis. The percentage of total variance explained by each axis in PCA is shown. Numbers from 1 to 6, represent the pathogens Aspergillus spp., B. fuckeliana, M. laxa, M. fructigena, P. expansum and P. digitatum, respectively.
Figure 6.
Classification of plant extracts according to colony growth of six postharvest fungi by PCA and cluster analysis. The percentage of total variance explained by each axis in PCA is shown. Numbers from 1 to 6, represent the pathogens Aspergillus spp., B. fuckeliana, M. laxa, M. fructigena, P. expansum and P. digitatum, respectively.
The activity of plant extracts on conidial germination clearly distinguishes three different groups (
Figure 7). Each one occupied very different ordinal spaces, indicating that inhibition differed substantially among the treatments.
Figure 7 shows that the principal components PC1 and PC2 accounted for 96.15% of the variation. Based on PCA and cluster analysis, there are three different groups: I DvA 6 (main inhibition), II DvA 1 to 5 (intermediate) and III FcR 1 to 6 (minor inhibition) (
Figure 7). The effectiveness on colony growth and conidial germination varied among the extracts and tested fungi based on PCA and cluster analysis.
Figure 7.
Classification of plant extracts according to conidia germination of six postharvest fungi by PCA and cluster analysis. The percentage of total variance explained by each axis in PCA is shown. Numbers from 1 to 6, represent the pathogens Aspergillus spp., B. fuckeliana, M. laxa, M. fructigena, P. expansum and P. digitatum, respectively.
Figure 7.
Classification of plant extracts according to conidia germination of six postharvest fungi by PCA and cluster analysis. The percentage of total variance explained by each axis in PCA is shown. Numbers from 1 to 6, represent the pathogens Aspergillus spp., B. fuckeliana, M. laxa, M. fructigena, P. expansum and P. digitatum, respectively.