Antifungal Activity of New Diterpenoid Alkaloids Isolated by Different Chromatographic Methods from Delphinium peregrinum L. var. eriocarpum Boiss

This paper aimed to investigate the potential antifungal influences of new alkaloids from Delphinium peregrinum L. var. eriocarpum Boiss. New Diterpenoid alkaloids Delcarpum (1), Hydrodavisine (4) and known alkaloids Peregrine (2), Delphitisine (3) were isolated by different chromatographic methods from the aerial parts of D. Peregrinum eriocarpum Boiss, which grows in Syria. The structures of alkaloids were proposed based on 1D NMR spectroscopy 1H-NMR, 13C-NMR, DEPT-135, DEPT-90, 2D NMR spectroscopy DQF-COSY, HMQC, EI-Ms mass spectrum, and IR spectroscopic measurements. The antifungal activity of the isolated alkaloids was evaluated against different dermatophyte fungal isolates compared with fluconazole. In the case of Peregrine (2) the minimum inhibitory concentrations(MICs) recorded 128–256, 32–64, and 32 for Epidermophyton floccosum, Microsporum canis, and Trichophyton rubrum, respectively, compared to 32–64, 16, and 32 μg/mL in the case of fluconazole, respectively. The MICs recorded on application of the four alkaloids mixture were 64, 32, and 16 in the case of E. floccosum, M. canis, and T. rubrum, respectively, which were significantly lower than that measured for each of the individual alkaloid and were compatible for fluconazole. In conclusion, MICs of the tested alkaloids showed a variable potential effect on the investigated fungal isolates. Peregrine (2) was the most effective alkaloid, however, the application of the mixture of alkaloids induced significant synergistic activity that was more pronounced than the application of individual ones.


Introduction
The genus Delphinium has been identified in many herbal species in Syria [1]. Delphinium is a medical herb used for the treatment of epilepsy, neurotoxic, and asthma [2,3]. The Delphinium alkaloids are currently under investigation in search for new analgesic, antiinflammatory drugs, muscular relaxation, anticonvulsant, pyramis lesion, and cardiac effects [3][4][5]. Moreover, a certain number of natural diterpenoid alkaloids have been reported to possess antiproliferative activities against various human cancer cell lines, indicating their great potential as new drugs for treating the corresponding cancers [6,7]. Due to their diterpenoid alkaloids components, Delphinium species have been considered as insecticidal potential [8]. Delphinium species have been used in folk medicine as a parasiticide and for the treatment of itches, skin eruptions. Delphinium was also used in the manufacture of dyes that are used to combat lice [3,9]. The plant D. peregrinum L. var. eriocarpum Boiss, which grows in Syria, has never been studied before. The first closest plant to it, called D. peregrinum var. elongatum Boiss, has been collected from Spain. C 19 -diterpenoid alkaloids (bicoloridine, dihydrogadesine, nudicaulidine, 13-acetylhetisinone, peregrine, peregrine alcohol, pergilone, delphiperegrine, and 14-O-acetylperegrine), norditerpenoid alkaloids (dehydrobicoloridine, bicoloridine alcohol, and peregrinine) and C 20 -diterpenoid alkaloids (hetisinone, hetisine, and atisinium chloride) have been isolated from this plant [10,11]. The second closest plant, called D. peregrinum has been collected from Turkey. C 19 -diterpenoid alkaloids (peregrine alcohol, pergilone, nudicaulidine, bicoloridine, peregrine, delphiperegrine) have been isolated from this plant [12]. Based on the aforementioned therapeutic effects, Delphinium alkaloids are expected to have antifungal effects. According to the available information, the literature has not sufficiently highlighted the expected antifungal effects of the new alkaloids isolated from D. Peregrinum L. Var. Eriocarpum Boiss in this study. This paper aimed to investigate the potential antifungal influences of new alkaloids isolated by different chromatographic methods from D. peregrinum L. var. eriocarpum Boiss ( Figure 1). insecticidal potential [8]. Delphinium species have been used in folk medicine as a parasiticide and for the treatment of itches, skin eruptions. Delphinium was also used in the manufacture of dyes that are used to combat lice [3,9]. The plant D. peregrinum L. var. eriocarpum Boiss, which grows in Syria, has never been studied before. The first closest plant to it, called D. peregrinum var. elongatum Boiss, has been collected from Spain. C19diterpenoid alkaloids (bicoloridine, dihydrogadesine, nudicaulidine, 13-acetylhetisinone, peregrine, peregrine alcohol, pergilone, delphiperegrine, and 14-O-acetylperegrine), norditerpenoid alkaloids (dehydrobicoloridine, bicoloridine alcohol, and peregrinine) and C20-diterpenoid alkaloids (hetisinone, hetisine, and atisinium chloride) have been isolated from this plant [10,11]. The second closest plant, called D. peregrinum has been collected from Turkey. C19-diterpenoid alkaloids (peregrine alcohol, pergilone, nudicaulidine, bicoloridine, peregrine, delphiperegrine) have been isolated from this plant [12]. Based on the aforementioned therapeutic effects, Delphinium alkaloids are expected to have antifungal effects. According to the available information, the literature has not sufficiently highlighted the expected antifungal effects of the new alkaloids isolated from D. Peregrinum L. Var. Eriocarpum Boiss in this study. This paper aimed to investigate the potential antifungal influences of new alkaloids isolated by different chromatographic methods from D. peregrinum L. var. eriocarpum Boiss ( Figure 1).

Chemistry
The alkaloids were isolated by column chromatography (C.C) on silica gel of (8.02 g) using a Et2O and Et2O-CHCl3 then CHCl3 and CHCl3/MeOH/NH3 step gradient, followed by further C.C and flash chromatography (F.C), then by preparative TLC. Purification of diterpenoid alkaloids was assigned by TLC, GC, and HPLC.

Chemistry
The alkaloids were isolated by column chromatography (C.C) on silica gel of (8.02 g) using a Et 2 O and Et 2 O-CHCl 3 then CHCl 3 and CHCl 3 /MeOH/NH 3 step gradient, followed by further C.C and flash chromatography (F.C), then by preparative TLC. Purification of diterpenoid alkaloids was assigned by TLC, GC, and HPLC. New alkaloids were determined by mass and NMR data, showed characteristic signals of C 19 -diterpenoid alkaloids and C 20 -diterpenoid alkaloids in their NMR spectra and characteristic fragmentation of such compounds in their mass spectrum [13,14]. The NMR spectra of Delcarpum (1). C 28 H 43 NO 8 gave signals at δ H 1.14 (3H, t, J = 6.99 Hz), δ C 11.9 (q) of an N-ethyl group, and a signal at δ H 0.85 (3H, s), δ C 25.2 (q) of methyl group, a signal at δ H 3.32 and 3.45 (3H, s) of two methoxy groups, and one methoxy group at δ H 3.17 (3H, m). The 13 C-NMR spectrum (Table 1) of Delcarpum (1) gave signals at δ C 48.5 (t) and δ C 25.2 (q) of an angular methyl group, and one methoxy group at δ C 48.6 (q) and at δ C 20.2 (q), 20.6 (q), 170.0 (s), and 170.3 (s). The 13 C-NMR spectrum of Delcarpum (1) contained only three signals up field from 81 ppm at δ C 32.8 (C-4), 47.6 (C-11), and 80.8 ppm (C-8) indicating the compound possessing is an aconitine-type C-19 diterpenoid alkaloids with a tertiary methoxy group at C-8 at δ C 48.6 (q) [4].
The other two methoxy groups were situated at C-1α and C-16β to account for the one-proton signals at δ H 3.22 (m, H-1β) and 3.11 (m, H-16α), which, in turn, gave one-bond connectivity with the methine carbon resonant at δ C 82.3 (d) and 91.4 (d) ppm, respectively, in the HMQC spectrum (Table 1). Two acetate groups were situated at C-6β and C-14α to account for the one-proton signals at δ H 5.29 (d, J = 7.53, H-6α) and 4.67 (t, J = 4.71 H-14β), which are connected with C-6, C-14, which showed a signal δ C 71.4 (d) and 75.1 (d) ppm, respectively, in the HMQC spectrum ( Table 1).
The methine carbon resonances at δ C 74.1 (d) ppm suggested the presence of the secondary hydroxyl group at δ H 3.52 (br s, OH) connected with C-15α in the molecule [10,11], and one proton of both protons connected with carbon C-12 gave a signal at δ H 2.50 (dd, J 1 = 3.17, J 2 = 12.6 Hz, H-12) in which C-12 gave a signal at δ C 28.2 (t) ppm, while one of the adjacent proton connected with C-13 gave a signal at δ H 2.32 (t, J = 5.54 Hz, H-13) in which C-13 gave a signal at δ C 36. 4  possessing is an aconitine-type C-19 diterpenoid alkaloids with a tertiary methoxy group at C-8 at δC 48.6 (q) [4]. The other two methoxy groups were situated at C-1α and C-16β to account for the one-proton signals at δH 3.22 (m, H-1β) and 3.11 (m, H-16α), which, in turn, gave one-bond connectivity with the methine carbon resonant at δC 82.3 (d) and 91.4 (d) ppm, respectively, in the HMQC spectrum (Table 1). Two acetate groups were situated at C-6β and C-14α to account for the one-proton signals at δH 5.29 (d, J = 7.53, H-6α) and 4.67 (t, J = 4.71 H-14β), which are connected with C-6, C-14, which showed a signal δC 71.4 (d) and 75.1 (d) ppm, respectively, in the HMQC spectrum ( Table 1).
The methine carbon resonances at δC 74.1 (d) ppm suggested the presence of the secondary hydroxyl group at δH 3.52 (br s, OH) connected with C-15α in the molecule [10,11], and one proton of both protons connected with carbon C-12 gave a signal at δH 2.50 (dd, J1  (Table 1) were in agreement with the proposed structure, and assignments were made by comparison with spectra of Peregrine (2) and 1 H-COSY and HMQC data (Table 1)  The mass spectrum (EIMS) of Delcarpum (1) has a peak at m/z 521, it represents the molecular ion M + which is corresponding to its molecular weight with formula C28H43NO8, the spectral region adjacent to the M-peak may be used to reveal substituent and functional groups, such as the base peak appears at m/z 491, which indicated the presence of methoxy group, which converted into formyl H2C=O, CH3O (M-31), ethyl (M-29), which are corresponding to the following peaks of m/z 490(3), 492(24), respectively. The mass spectrum also gave the fragments of m/z -CH2OCH3 45, CH3-C=O 43 The mass spectrum (EIMS) of Delcarpum (1) has a peak at m/z 521, it represents the molecular ion M + which is corresponding to its molecular weight with formula C 28 H 43 NO 8 , the spectral region adjacent to the M-peak may be used to reveal substituent and functional groups, such as the base peak appears at m/z 491, which indicated the presence of methoxy group, which converted into formyl H 2 C=O, CH 3 O (M-31), ethyl (M-29), which are corresponding to the following peaks of m/z 490(3), 492(24), respectively. The mass spectrum also gave the fragments of m/z -CH 2 OCH 3 45, CH 3 -C=O 43 Peregrine (2), a colorless crystalline solid, gave a molecular ion at m/z 463 (M + , 3) in its EIMS, accounting for a molecular formula of C 26 H 40 NO 6 . The chemical structure of compound (2) was established according to its IR and NMR spectra (Table 1) [10,11,15].  , H-20). The 13 C-NMR spectrum showed twenty-one signals, including one methyl, eight methylene, eight methine, and four quaternary carbons C-4, C-8, C-10, and C-16 that gave signals at δ C 35.7, 44.1, 53.4, and 154.1 ppm, respectively ( Table 1) (3) has a peak at m/z 327 (54%) represents the molecular ion M + , which is corresponding to its molecular weight and a molecular formula of C 21 H 29 NO 2 . All that data can be satisfactorily supported by the structure of Delphitisine (3). The closest type is Hetisine-type, which is one of the most complex entries in the atisane-class [10,11,16,17].  (3) has a peak at m/z 327 (54%) represents the molecular ion M + , which is corresponding to its molecular weight and a molecular formula of C21H29NO2. All that data can be satisfactorily supported by the structure of Delphitisine (3). The closest type is Hetisinetype, which is one of the most complex entries in the atisane-class [10,11,16,17].  Table 1). The group (C3N + H) has been shown by signals at δC (C6: 63.6, C19: 59.4, C20: 66.3 ppm) and a hydrogen atom (1H, 3.74 ppm). 13 C-NMR spectrum showed twenty signals, including one methyl, seven methylene, eight methine, and four quaternary carbons C-4, C-8, C-10, and C-16 that gave signals at δC 35.4, 36.3, 54.2, and 154.2 ppm, respectively (Table 1). After studied COSY and HMQC experiments (in CD3OD) it found the partial structures (A), (B), (C), and (D) (Figures 4 and S3a-h), and after comparing the 13 C-NMR of Hydrodavisine (4) with its of Delphitisine (3) and of C20-diterpenoid alkaloids [5,10,11]. We made sure that this compound has the same skeleton of Delphitisine (3), losing a secondary carbon atom. In addition to that, the mass spectrum of Hydrodavisine (4) (EIMS) the molecular weight M + = 331 is corresponding to its molecular formula of [C20H29NO3], a peak at m/z 314 (96%) represents the molecular ion (M-OH) + . The peak at m/z 348 was for the fragment [M + OH] + , and the peak m/z 349 was for the fragment (M + H2O) + . The peak in 1 H-NMR 4.89 ppm for OH hydrogen bonds with water molecules [15,18].  Table 1). The group (C 3 N + H) has been shown by signals at δ C (C 6 : 63.6, C 19 : 59.4, C 20 : 66.3 ppm) and a hydrogen atom (1H, 3.74 ppm). 13 C-NMR spectrum showed twenty signals, including one methyl, seven methylene, eight methine, and four quaternary carbons C-4, C-8, C-10, and C-16 that gave signals at δ C 35.4, 36.3, 54.2, and 154.2 ppm, respectively (Table 1). After studied COSY and HMQC experiments (in CD 3 OD) it found the partial structures (A), (B), (C), and (D) (Figures 4 and S3a-h), and after comparing the 13 C-NMR of Hydrodavisine (4) with its of Delphitisine (3) and of C 20diterpenoid alkaloids [5,10,11]. We made sure that this compound has the same skeleton of Delphitisine (3), losing a secondary carbon atom. In addition to that, the mass spectrum of Hydrodavisine (4) (EIMS) the molecular weight M + = 331 is corresponding to its molecular formula of [C 20 H 29 NO 3 ], a peak at m/z 314 (96%) represents the molecular ion (M-OH) + . The peak at m/z 348 was for the fragment [M + OH] + , and the peak m/z 349 was for the fragment (M + H 2 O) + . The peak in 1 H-NMR 4.89 ppm for OH hydrogen bonds with water molecules [15,18].
NMR of Hydrodavisine (4) with its of Delphitisine (3) and of C20-diterpenoid alkaloids [5,10,11]. We made sure that this compound has the same skeleton of Delphitisine (3), losing a secondary carbon atom. In addition to that, the mass spectrum of Hydrodavisine (4) (EIMS) the molecular weight M + = 331 is corresponding to its molecular formula of [C20H29NO3], a peak at m/z 314 (96%) represents the molecular ion (M-OH) + . The peak at m/z 348 was for the fragment [M + OH] + , and the peak m/z 349 was for the fragment (M + H2O) + . The peak in 1 H-NMR 4.89 ppm for OH hydrogen bonds with water molecules [15,18].

Biology
Antifungal Activity MIC of the tested alkaloids showed a different potential effect on the investigated fungal isolates. Peregrine was the most effective where the MIC recorded 128-256, 32-64, and 32 for Epidermophyton floccosum, Microsporum canis, and Trichophyton rubrum, respectively, compared to 32-64, 16, and 32 µg/mL in the case of fluconazole. Alkaloids extracted from the plant have been known to have important characteristics with biochemical, pharmacological, and medical effects in living organisms [19,20]. The investigators isolated three C 19 -diterpenoid alkaloids: Delbrunine, delbruline, and delbrusine, from Delphinium brunonianum and they indicated that these compounds have an antibacterial effect against Escherchia coli, Staphylococcus aureus, Pseudomonas aureginous, Salmonella flexinarie, and Bacillus subtilis. Sometimes, the use of individual bioactive compounds extracted from a particular plant does not induce the predicted inhibitory effects compared to its original synergistic combination with other associate candidates. The MIC and MFC of the mixture of tested alkaloids were significantly more pronounced than the application of individual ones. The MICs recorded on the application of the mixture of the four alkaloids were 64, 32, and 16 in the case of E. floccosum, M. canis, and T. rubrum, respectively, which was significantly lower than that measured for each of the individual alkaloids and were compatible for fluconazole as reference standard drug (Table 2). Hemaiswarya et al. [21] indicated that several plant extracts have shown synergistic activity against microorganisms. This review designates in detail the observed synergy and mechanism of action between natural products including flavonoids and essential oils and synthetic drugs in combating microbial infections. The mode of action of combination differs significantly from that of the same drugs acting individually, hence, isolating a single component may lose its therapeutic importance. The mean value of replicates for MIC and MFC was calculated after considering the standard deviation for the data.