Chemical composition of A. viridiflora methanol extract
The major phenolic compounds found in genus Alchemilla belonged to the ellagitannins (5-8%), principally the dimers agrimoniin (3.5-3.8%) and laevigatin F (0.9%) and the monomer pedunculagin (1.2%) [25]. Ellagitannins are hydrolysable tannins esterified with hexahydroxydiphenic acid (HHDP) and a most often glucose. Typical neutral losses of ellagitannins during MS fragmentation are galloyl (152 Da), gallic acid (170 Da), hexahydroxydiphenic acid, HHDP, (302 Da), galloylglucose (332 Da), HHDP glucose (482 Da), and galloyl-HHDP-glucose (634 Da) residues and characteristic UV-vis spectra at 254 nm and 360-368 nm. Flavonoids are also often found in the genus Alchemilla. Those are dominantly derivatives of flavonols, quercetin and kaempferol. Quercetin and its derivate have absorption maximum at 354 nm and characteristic MS fragmentation ion at m/z 301 (negative mode), while kaempferol and its derivate have λmax at 348 nm and characteristic MS fragment ion at m/z 285 (negative mode) [11, 12, 14, 26, 27]. Phenolic acids, such as gallic, 3, 4-dixydroxy-benzoic acid, chlorogenic and caffeic acidare also commonly present [28].
Preliminary studies have shown the presence of polyphenolic compounds (233.41 ± 3.29 µg GA/mg of dry extract), flavonoids (0.30 ± 0.05%) and tannins (3.74% ± 0.98) in methanol extract of A. viridiflora. LC-MS analysis leads to the identification of 20 compounds in methanol extract, mainly ellagitannins and flavonoid glycosides (Fig. 1, Table 1). This was the first time that chemical analysis of A. viridiflora was done. Also, some compounds were identified for the first time in Alchemilla species.
Table 1 Results of LC-MS chemical analysis of methanol extract Alchemilla viridiflora
Peak
|
Rt (min)
|
Molecular formula¶
|
Λmax (nm)
|
MW
|
[M − H] − (m/z) (100 V)
|
MS data (m/z) (250V)
|
Compound name
|
1
|
3.567
|
C20H18O14
|
236, 314
|
482
|
481
|
301, 275
|
HHDP-hexoside†
|
2
|
6.138
|
C34H24O22
|
232
|
784
|
783
|
481, 301
|
Pedunculagin I isomer†
|
3
|
9.188
|
C34H24O22
|
232
|
784
|
783
|
481, 301
|
Pedunculagin I isomer
|
4
|
12.247
|
C27H22O18 C27H28O18
|
222, 274 258, 352
|
634 640
|
633 639
|
463, 301
|
Galloyl – HHDP-glucose† Quercetin-hexoside- glucuronide†
|
5
|
14.140
|
C13H8O8
|
278, 360
|
292
|
291
|
247, 219, 190
|
Brevifolin carboxylic acid†
|
6
|
16.256
|
C34H26O22
|
218, 274
|
786
|
785
|
755, 301
|
Tellimagrandin I§
|
7
|
18.865
|
C68H48O44
|
230, 278
|
1568
|
783
|
1265, 1103, 935, 631, 301
|
Sanguiin H-10 isomer†
|
8
|
20.945
|
C41H28O26
|
224, 278
|
936
|
935
|
633, 301
|
Galloyl-bis-HHDP-glucose†
|
9
|
22. 167
|
C68H48O44
|
230, 278
|
1568
|
783
|
1265, 1103, 935, 631, 301
|
Sanguiin H-10 isomer†
|
10
|
23.906
|
C41H30O26
|
222, 280
|
938
|
937
|
433, 301
|
Tellimagrandin II§
|
11
|
25.544
|
C21H18O13
|
254, 308, 354
|
478
|
477
|
301
|
Miquelianin‡
|
12
|
26.383
|
C82H54O52
|
254, 306 sh, 368
|
1870
|
934
|
1567, 1265, 1085, 934, 633, 301
|
Agrimoniin†
|
13
|
28.121
|
C19H14O12
|
256, 354
|
434
|
433
|
301
|
Ellagic acid pentose†
|
14
|
29.331
|
C41H21O26
|
220, 280
|
940
|
939
|
787, 769, 635
|
Pentagalloylglucose†
|
15
|
30.965
|
C22H20O13
|
252, 356
|
492
|
491
|
315, 301, 275
|
Quercetin methyl ether glucuronide†
|
16
|
33.735
|
C23H22O13
|
254, 358
|
506
|
505
|
329, 301, 269
|
Quercetin dimethyl ether glucuronide†
|
17
|
36.518
|
-
|
n.d.
|
712
|
711
|
665, 503, 465
|
Formate aduct of triterpene acid hexoside†
|
18
|
37.173
|
-
|
n.d.
|
710
|
709
|
663, 501
|
Formate aduct of triterpene acid hexoside†
|
19
|
37.592
|
C30H26O13
|
226, 266, 314
|
594
|
593
|
447, 283
|
Tiliroside‡
|
20
|
37.933
|
-
|
n.d.
|
696
|
695
|
649, 487
|
Formate aduct of triterpene acid hexoside†
|
HHDP - hexahydroxydiphenic acid.
¶source PubChem®
†tentatively identified by comparing with literature data
‡identified comparing with commercial standard
§identified comparing with previously isolated compound
Peak 1 produced a [M− H]− ion at m/z 481 and generated fragment ion m/z 301 [M − 180 − H]− (loss of hexose) corresponding to an HHDP residue, and m/z 275 by decarboxylation of the HHDP moiety [29]. This compound has been identified as HHDP-hexoside. Monohexosides of HHDP were identified before in Rosaceae family, in leaves methanol extract of wild blackberries, Rubus grandifolius L. [30], but were never identified or isolated from Alchemilla species.
Peaks 2 and 3 correspond to isomeric compounds, with the [M − H]− ion at m/z 783, yielding main fragment ions at m/z 481 [M − 302 − H]− (loss of HHDP) and 301 [M − 482 − H]− (loss of HHDP-glucose), whose fragmentation pattern corresponds to a bis-HHDP-glucose structure presumably pedunculagin I isomers, which were previously reported in acetone-water extract of aerial parts of both A. vulgaris and A. mollis, as well as in methanol extract of aerial parts of A. persica Rothm. [13, 31].
Peak 4 had [M − H] − at both m/z 633 and 639 with main fragments at m/z 463 [M − 170 − H] − (loss of gallic acid) and 463 [M − 176 − H] − (loss of glucuronide unit) and m/z 301 [M − 332 − H]− (loss of galloylglucose) and 301 [M − 338 − H]− (loss of hexoside-glucuronic unit) and. The same mass [M -H] − at m/z 633 can be seen in m-galloyl-HHDP-glucose, but this compound has a main fragment at m/z 481 [M − 152 − H] − (loss of galloyl moiety) which suggests that the galloyl unit is probably bonded via an m-depside bond, and not attached directly to the glucose core. On the contrary, loss of gallic acid in Peak 4 indicates that the galloyl unit is attached directly to the glucose [27]. Accordingly, peak 4 is identified as galloyl-HHDP-glucose (presumably corilagin isomer). In the case of compound with ion at m/z 639 it clearly indicates that the compound can be identified as quercetin-hexoside- glucuronide. These compounds were both previously identified in A. vulgaris and A. mollis as well as A. persica [13, 31].
Peak 5 exhibited the [M − H] − ion at m/z 291 and fragment ions at m/z 247 [M − 45 − H] − (loss of carboxylic acid) which corresponds to brevifolin, ion m/z 219 [M − 73 − H] − which corresponds to the loss of C = OCOOH and m/z 191, which are characteristic fragment ions for brevifolin carboxylic acid according to literature. This compound has never been identified in Alchemilla species before. Brevifolin carboxylic acid is considered as an end product of ellagitannin hydrolyses, and it can be commonly found in whether fruits, leaves, flowers or heartwood of pomegranate, Punica granatum [32]. Nevertheless, this ellagitannin has previously been reported in species from Rosaceae family. It has been isolated from raspberry (Rubus idaeus L.) juice and whole plant of Duchesnea indica (Andrews.) Focke. [33, 34].
Peak 6 [M − H] − ion at m/z 785 with fragment 392 [M - 2H] 2− was reported for the first time in Alchemilla species. This compound was identified as tellimagrandin I using a standard compound previously isolated from flowers of Filipendula vulgaris Moench. [35].
Peaks 7 and 9 were tentatively identified as sanguiin H-10 isomers, previously reported in Alchemilla species (A. vulgaris, A. mollis and A. persica) with fragment ion at m/z 783. This dimeric ellagitanninis composed of galloyl-bis-HHDP-glucose (m/z 935) and galloyl-HHDP-glucose (m/z 783). The proposed fragment ions at m/z l265 [M-302-H] − (loss of HHDP), 1103 [M− 464− H] − (loss of HHDP-glucose), 933 [M−634− H] − (loss of galloyl-HHDP-glucose), 631 [M-936-H] − (loss of HHDP-glucose-galloyl-HHDP) and 301 [M− 1266− H] − (loss of galloyl-HHDP-glucose-galloyl-HHDP-glucose) [13, 31].
Peak 8 [M − H] − ion at m/z 935 and fragment ions at m/z 783 [M − 152 − H] − (loss of galloyl unit), 633 [M − 302 − H]− (loss of HHDP) and 301 [M − 634 − H]− (loss of galloyl-HHDP-glucose) is identified as galloyl-bis-HHDP-glucose. This compound is a commonly present unit in structure of many ellagitannins, such as dimeric ellagitannin agrimoniin, and it has been previously identified in Alchemilla vulgaris and A. mollis species [13].
Peak 10 is identified first time in Alchemilla species as tellimagrandin II using a standard compound previously isolated from flowers of Filipendula vulgaris [M − H] − ion at m/z 937 with fragment ions m/z 767 [M – 170− H] – (loss of galloyl unit), m/z 468 [M-2H]2– and 301[M-634-H] – (loss of galloyl-HHDP-glucose) [36].
Peak 11 corresponds to the mass spectra of used standard substance quercetin-3-O-β-glucuronide (miquelianin) with [M − H] − ion at m/z 477, with a fragment ion at m/z 301 [M − 176 − H] − (loss of glucuronide), which corresponds to quercetin. As mentioned before, miquelianin is a commonly found flavonoid in Alchemilla species.
Peak 12 corresponds to agrimoniin, common ellagitannin is Alchemilla species, since it has been recognized as a marker compound of the Rosaceae family [13]. It showed a [M−H]− ion at m/z 1869 and fragment ions at m/z 1567 [M− 302− H]– (loss of an HHDP unit), 1265 [M− 604− H]– (loss of bis-HHDP), 1085 [M− 784− H]– (loss of bis-HHDP-glucose) and the main fragment at m/z 935 [M− 2H]2–, corresponding to one galloyl-bis-glucose unit, followed then by fragmentation ions at m/z 633 (935 − 302 , loss of HHDP unit) and 301 (633 − 332, loss of galloyl-glucose residue) [37].
Peak 13 has [M − H] − ion at m/z 433 and fragment ion at m/z 301 [M − 132 − H] − (loss of a pentose), corresponding to the both ellagic acid and quercetin. As mentioned before, ellagic acid and it derives show characteristic UV spectra with λmax at 254 nm and 360-368 nm, while quercetin and its glycosides are with absorption maximum at about 354 nm. Hence, this compound is proposed to be ellagic acid pentose. Ellagic acid is a common constituent of Alchemilla species, while its conjugates are reported for the first time in this paper. These metabolites can be found in other species of Rosaceae family, such as in acetone extract of strawberry fruits (Fragaria x ananassa) [27].
Peak 14 shows [M− H] − at m/z 939 and a fragment ion at m/z 769 [M− 170− H]− corresponding to loss of gallic acid moiety. In addition, the appearance of fragment ions at m/z 787 and 635 corresponds to the loss of the first galloyl moiety [M− 152− H] −, and the second one [M− 304− H] − respectively, thus this compound can be interpreted as pentagalloylglucose, found in methanol extract from leaves of Eucalyptus globulus Labill. [38].
Peak 15 and 16 exhibits [M − H]− ion at m/z 491 and 505, respectively, with fragment ions at m/z 329 and m/z 315 [M − 176 − H]− which clearly indicates the loss of glucuronide unit, while the fragment of m/z 301 in both peaks indicates to presence of quercetin aglycone ([M − 190 − H]− loss of glucuronide and methylene moiety; [M − 204 − H]− loss of glucuronide and two methylene units). The proposed compounds are tentatively identified as quercetin methyl ether glucuronide, previously found in Alchemilla species [13], and quercetin dimethyl ether glucuronide. This quercetin conjugate has never been reported neither in Alchemilla species or Rosaceae family.
Peaks 17, 18 and 20 exhibit an [M− H] − ion at m/z 711, 709 and 695, respectively. This form of fragmentation may correspond to triterpene components found in raspberry, Rubus ideus [39]. Namely, all the compounds show proposed loss of formate [M-46-H] −, giving fragments at m/z 665, 663 and 649, respectively, followed by loss of hexose moiety [M− 162− H] − to form the fragments at m/z 503, 501 and 487, respectively. These fragments are presumably aglycones of pentacyclic triterpenoids, ursane type (asatic and madecassic acid) [40] and olean type (serjanic acid) [41]. Therefore, these peaks could be tentatively identified as formate addicts of triterpene acid-O-hexoside. These compounds were not identified in Alchemilla species before. Nevertheless, triterpenes, sucha as ursolic acid, 2-a-hy-droxyursolic acid, tormentic acid, euscophic acid, and oleanolic acid, have been identified in aerial parts of A. vulgaris, A. alpine L. and , A. faroënsis (Lange) Buser [42].
Peak 19 exhibits an [M− H] − ion at m/z 593 and a fragment ion at m/z 285 [M− 308− H] − (loss of a coumaroyl glucoside moiety) which corresponds to kaempferol, in addition to fragment ions at m/z 447 [M− 146− H] − (loss of p-coumaroyl) which corresponds to kaempferol glucoside. This compound is identified based on the comparison of its UV and an MS spectrum to those of commercially available standard as kaempferol-3-O-(6-p-coumaroyl)-glucoside, tiliroside. This is the first report of presence of tiliroside in tested Alchemilla species. Tiliroside has been isolated before from other Alchemilla species, such as from methanol extracts aerial parts of A. vulgaris, A. barbatiflora Juz. and A. mollis [10, 12, 43].
ACE inhibitory activity
Molecules obtained from various plant isolates have gained great interest as ACE inhibitors recently. The most promising compounds based on their structure difference could be divided in tannins, flavonoids, essential oil [5, 44]. Although structurally different, all these compounds have in common presence of functional groups which serve as hydrogen (H) bond acceptors or donors, such as phenolic and carboxylic. Different concentration ranges of the A. viridiflora methanol extract rich in flavonoids and ellagitannins and miquelianin standard were evaluated in vitro for ACE activity inhibition. The results of ACE inhibitory activity of tested sample at the concentration range of 0.0016–5 mg/mL are presented in Fig. 2 and ACE inhibitory activity of miquelianin standard at the concentration range of 0.00032–1 mg/mL in Fig. 3.
The dose-dependent activities of A. viridiflora and miquelianin were observed in the tested concentration ranges. The result obtained from in vitro investigation of ACE inhibitory activity of methanol extract of A. viridiflora showed IC50 of 2.51 ± 0.00 µg/mL compared to IC50 of 2.59 ± 0.00 µg/mL for miquelianin standard. According to the manufacturer of ACEKit-WST (Dojindo, Japan), IC50 of Alacepril and Captopril are 3.62 μM and 2.14 nM, respectively. Our extract had lower IC50 than standard substance miquelianin, whose activity was significantly lower than captopril, but in range with alacepril, potent synthetic ACE inhibitor.
The result obtained from molecular docking simulation study showed that 3 out of 20 compounds from methanol extracts obtained from A. viridiflora, formed more stable complexes with amino-acid residues in receptor binding site than lisinopril, well known synthetic ACE inhibitor (positive control) under the same experimental conditions (Table 2). Further interaction analysis revealed that all complexes were stabilized by conventional H bond interactions between receptor and selected ligands. Moreover, complexes were additionally stabilized Van der Waals and π-interactions (Fig. 4).
Table 2 Summarized docking simulation results for identified constituents of studied Alchemilla viridiflora which formed more stable complexes than positive control lisinopril (7.564 kcal/mol)
Compound
|
Pubchem ID
|
Bind.energy [kcal/mol]
|
Hydrogen bonds contacting receptor residues
|
Tiliroside
|
5320686
|
-10.750
|
Glu 143, Ala 356, His 513
|
Ellagic acid pentose
|
5487461
|
-10.364
|
Ala 354, Lys 511, His 513, Tyr 520
|
Galloyl-HHDP-glucose
|
503250
|
-10.258
|
Gln 281, Ala 354, Cys 370, Glu 384, Asp 453
|
Miquelianin
|
57331105
|
-9.894
|
Thr 166, Asn 277, Gln 281, Thr 282, Glu 384
|
Tellimagrandin I
|
442690
|
-9.678
|
Asn 70, His 353, Asp 358, Tyr 360, Glu 411, Arg 522
|
Brevifolin carboxylic acid
|
9838995
|
-8.576
|
Gln 281, Asp 411
|
Fig. 4: 2D schematic diagrams of key receptor amino acid residues interactions with selected ligands from A. viridiflora sample: a) brevifolin carboxylic acid, b) ellagic acid pentose, c) galloyl-HHDP-glucose, d) miquelianin, e) tellimagrandin I and f) tiliroside
Computational docking results showed that tiliroside, ellagic acid pentose and galloyl-HHDP glucose exhibited even better binding affinity for ACE active site than miquelianin, which ACE activity was confirmed by an in vitro assay. The best affinity for receptor binding site was registered for tiliroside, flavonoid which demonstrated significant antihypertensive properties in a previously reported study [45].
Investigation of potential inhibitory effects on ACE activity of 11 Cuphea spp. crude extracts along with pure compounds showed that polyphenol miquelianin exhibited inhibitory activity comparable to captopril, well known ACE inhibitor which was the case in our study also [46].
Interactions with amino acid residues: Glu 162, His 353, Ala 354, Asp 377, Glu 384, Lys 511, His 513, Tyr 520, Tyr 523 in ACE active site is considered as crucial for ACE enzyme activity inhibition [47]. Tiliroside, tellimagrandin I, galloyl-HHDP and ellagic acid pentose from A. viridiflora methanol extract stabilized the most favorable molecule orientation through conventional H bonds interactions with above mentioned residues.