|Year : 2009 | Volume
| Issue : 2 | Page : 43-52
Antifouling Alkaloids from Crinum augustum (Amaryllidaceae)
John Refaat1, Ahmed Abdel-Lateff1, Mohamed S Kamel1, Ahmed A Ali2, Mahmoud A Ramadan2, Tatsufumi Okino3, Yasuyuki Nogata4
1 Pharmacognosy Department, Faculty of Pharmacy, Minia University, 61519 Minia, Egypt
2 Pharmacognosy Department, Faculty of Pharmacy, Assiut University, Egypt
3 Faculty of environmental earth science, Hokkaido University, Sapporo, 060-0810, Japan
4 Central Research Institute of Electric Power Industry, Biology Department, 1646 Abiko, Chiba 270-1194, Japan
|Date of Submission||02-Jan-2009|
|Date of Acceptance||26-Feb-2009|
|Date of Web Publication||2-Jan-2010|
Pharmacognosy Department, Faculty of Pharmacy, Minia University, 61519 Minia
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Fractionation and purification of the ethanolic extract of the bulbs of Crinum augustum Rox. (Amaryllidaceae) cultivated in Egypt yielded five alkaloids 6-methoxy-crinamine (1) , crinamine (2) , buphanisine (3) , ungeremine (4) , and hippadine (5) ; two fatty acid derivatives: myristic acid ethyl ester (6) and palmitic acid ethyl ester (7) ; four terpenoidal and steroidal compounds: ursolic acid (8) , β-sitosterol-O- β glucopyranoside (9) and mixture of β-sitosterol (10) and stigmasterol (11) . The structures of all compounds were determined by interpretation of their spectroscopic data; 1D ( 1 H and 13 C), 2D (HSQC, COSY, DQF, NOE and HMBC) NMR; MS and UV analyses. The compounds (1 -4) and (6-8) were tested towards biofouling activity using larvae of barnacle Balance amphitrie. Significant activities of 1, 2 and 3 with EC 50 1.8, 1.2 and 0.75 μg/mL respectively, were observed.
Keywords: Crinum augustum, Amaryllidaceous, anti-fouling, natural products
|How to cite this article:|
Refaat J, Abdel-Lateff A, Kamel MS, Ali AA, Ramadan MA, Okino T, Nogata Y. Antifouling Alkaloids from Crinum augustum (Amaryllidaceae). Phcog Res 2009;1:43-52
|How to cite this URL:|
Refaat J, Abdel-Lateff A, Kamel MS, Ali AA, Ramadan MA, Okino T, Nogata Y. Antifouling Alkaloids from Crinum augustum (Amaryllidaceae). Phcog Res [serial online] 2009 [cited 2021 Apr 12];1:43-52. Available from: http://www.phcogres.com/text.asp?2009/1/2/43/58131
| Introduction|| |
Amaryllidaceae is a big family and contains about 90 genera and 1310 species  . They occur naturally throughout the tropics, subtropics and warm temperate regions of the world in Asia, Australia, Africa and America  . Members of Amaryllidaceae attract considerable attention due to various medicinal properties such as anti-tumor, immunostimulant, analgesic, antiviral, anti-malarial, antibacterial and antifungal activities. They are subjected to chemical, cytological and pharmacological investigations due to their richness in pharmacologically active alkaloids  such as lycorine, a principal alkaloid possessing a strong antiviral activity , and galanthamine which has anti-cholinesterase activity  that accounts for its use in Alzheimer's disease  . The genus Crinum is productive and well known with its diversity of chemical and biological activities , . Settlement of higher organisms such as macroalgae and invertebrates may threaten the survival of individuals of benthic invertebrates. Therefore, benthic invertebrates have developed various defense systems against biofouling, such as biofilm  . Biofouling on ships hulls, offshore structures or aquaculture equipment is a major global economic and technical problem. The organotin compounds have until recently been widely used for biofouling control, but after 2008 their use will be prohibited worldwide, Therefore, the development of environmentally safe antifouling substances is urgently needed  . Finally, the main task of the current paper is the discovering of new natural products with biological activity and/or novel chemical structures from Crinum augustum, particularly those having ecoenvironmental activity such as anti-fouling. Thus, successive fractionation of the ethanolic extract of Crinum augustum by vacuum liquid chromatography (VLC) over normal silica, yielded 1-11 [Figure 1]. The effect of compounds 1, 2 and 3 on settlement and variability of cyprids [Table 1] and [Figure 2], with EC 50 ; 1.8, 1.2 and 0.75 μg /mL respectively. CuSO 4 (EC 50 = 0.23 μg/mL) was used as a positive control.
| Materials and Methods|| |
General procedures- Melting points were measured using Stuart Scientific (SMPI) melting point apparatus and were uncorrected. UV spectra were determined using Spectronic® Genesys TM 2PC UV spectrophotometer. 1 H and 13 C-NMR spectra were recorded on a JEOL-JNM-EX-400 spectrometer (400 MHz for 1H and 100 MHz for 13 C, respectively). ESIMS data were obtained with a JEOL JMS-700T mass spectrometer. Pre-coated silica gel 60 GF254 plates (E. Merck) were used for TLC. The compounds were detected by UV absorption at λmax 254 and 366 nm followed by spraying with anisaldehyde/H 2 SO 4 reagent and heating at 110 °C for 1-2 min. All solvents were distilled prior to use.
The bulbs of Crinum augustum Rox. were collected from the farm of Faculty of Pharmacy, Assuit University, Egypt. The plant material was kindly identified by Prof. Dr. A. Fayed, Professor of plant taxonomy at Assuit University. A voucher specimen (C1) was deposited in herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Minia University, Minia, Egypt.
Extraction and isolation
The air-dried powdered bulbs (4 Kg) were extracted by 95% EtOH (3 X 5 L), and evaporated under reduced pressure into viscous extract (795 g). The solvent-free residue (795 g) was partitioned between 5% aqueous HCI and CHCl 3 (1:1). The CHCl 3 fraction (F001, 487 g). The mother liquor was rendered alkaline by 5% NH40H and successively extracted with CHCl 3 and EtOAc, yielded F002 (10.3 g) and F003 (11.4 g), respectively. The solution (mother liquor) was saturated with Na2CO3 and then re-extracted with CHCl 3i resulted in F004 (1.32 g). Finally, the aqueous mother liquor was concentrated to give fraction F005 (201.5 g). F001 (20 g) was fractionated on NP-silica (100 x 5 cm, 500 g, 60120 mesh, E-Merck) employing gradient elution using pet. ether- EtOAc (98:2; 95:5; 90:10; 70:30; 50:50, 0:100), (1.35, 4.0, 0.7, 2.55, 5 and 3.53 g) and washed with MeOH (1.2 g), 250 mL each of fraction. The column was monitored by TLC, the similar fractions were pooled into 14 pools. F012 was fractionated on NP-silica employing gradient technique using pet. ether-CHCl 3 (99:1, 98:2, 97:3) and then purified on NP-silica, using pet. ether CHCl 3 (99:01) to yield 6 (12.7 mg) and pet. ether-CHCl 3 (99:01) to yield 7 (199 mg). F0015 was purified on NP-silica, using gradient pet. ether EtOAc (97:3, 95:5 and 93:7), led to isolation of 5 (20 mg), re-crystallized by MeOH. F016 fractionated on NP-silica, employing pet. ether-EtOAc (95:5, 93:7, 90:10, 88:12) resulted in mixture of (10 and 11) (105 mg) and 8 (26.5 mg). F0111 was purified by NP-silica, using CHCl 3 -MeOH (99:1, 98:2, 97:3, 95:5, 93:7) to yield 9 (26 mg). F0113 was purified by using alkaline silica NP-silica, and employing pet. ether EtOAc (30:70, 25:75, 20:80, 10:90) and further purification on NP-silica using CHCl 3 -MeOH (98:2), yielded 1 (39 mg). F0114 was purified by using NP-silica, and employing pet. ether-EtOAc (30:70, 20:80, 10:90) and further purification on NP-silica using CHCl 3 -MeOH (98:2) yielded 3 (34.5 mg). F02 fractionated on NP-silica (100 x 5 cm, 310 g, 60-120 mesh, E-Merck), employing pet. ether-EtOAc (90:10; 70:30 ; 50:50; 25:75; 0:100) (0.1, 0.5, 0.5, 1.6, 2.5g, respectively) and washed with EtOAc-MeOH (50:50 and 0:100) (2.3 and 1.0 g), led to F021-27. F024 was fractionated on alkaline NP-silica using pet. ether CHCl 3 (40:60), yielded 2 (60 mg). F004 was fractionated on alkaline NP-silica (30 x 2 cm 30 g, 60-120 mesh, E-Merck), elutied with pet. ether-EtOAc (75:25, 50:50, 0:100) (20, 30, 80 mg, respectively), and washed with EtOAc MeOH (80:20 and 0:100) (100, 340 mg) yielded 4 (42 mg). F045 was fractionated on NP-silica (100 x 5, 340 g, 60-120 mesh, E-Merck) eluted with pet. ether EtOAc ( 70:30, 10:90, 0:100) yielded 4 (42 mg).
| Biological Activity|| |
The culture of Cyprid larvae has been reported previously  . The samples were dissolved in MeOH and aliquots were pipetted into 24-well polystyrene tissue culture plates and air-dried. Two mL of filtered seawater diluted to 80% by deionized water (80% filtered seawater) and 4-8 cyprids (2-3 days fold) were added to each well. Each level of the experiments was carried out with four wells (10-18 cyprids). The plates were kept in the dark at 25°C and the number of larvae that attached, metamorphosed, dried or did not settle, was counted after 48 H , , cyprids that did not move had extended appendages and did not respond after a light touch by metal were regarded as dead. The assay of seven compounds and controls was repeated 5 times with different batches of larvae. Each concentration was replicated 3-5 times. Normality of the distribution was verified with Shapiro-Wilk's test. When necessary, the percentages of settled and dead larval were analyzed after arcsine transformation. ANOVA followed by Dunnet's comparison test was used for multiple comparisons of treatment means with control. The antifouling activity of the compounds was expressed as ED 50 values. The ED 50 values were calculated by prohibt analyses. A non toxic solution was defined as one in which cyprids did not settle but remained alive after 48 h.
| Results and Discussion|| |
This class of compounds has been tested for the first time for their anti-fouling activity. There is no previous publication of antifouling activity based on a computer survey. This paper represents the first report of alkaloids with anti-fouling activity which opens the gate to the other researcher to find new antifouling activity from terrestrial natural product research.
Positive HR-ESI-MS of 1 revealed a molecular ion peak at m/z 332.14 [M+H] + to have molecular formula C 18 H 21 N0 5 . Decoupled 13 C and DEPT NMR spectra of 1 showed eighteen resonances [Table 3]. Eight of eighteen elements of unsaturation, as indicated by the molecular formula of 1 , could be attributed to four carbon-carbon double bonds and the molecule thus, has five rings. The NMR spectroscopic data further enabled all but one hydrogen atom of 1 to be attached to carbons; hence it was evident that the remaining one is present in the molecule as hydroxyl function. UV spectral data of 1 , exhibited three maxima absorptions (λmax 292, 241 and 205 nm), indicating the presence of a methylenedioxy-substituted benzene ring  revealing the presence of crinine nucleus. Interpretation of the 2D NOESY correlations is simplified assignment of the two pararomatic proton to be H-10 and H-7, respectively. From this spectral data, it was clear that H-10 (δH 6.9) has correlation with H-1 and H-7 (δH 6.71) is correlated with H-6. From 1 H NMR spectral data of 1 , four characteristic signals, two of them indicates olefinic protons δH 6.47 (d, J = 10.5 Hz, H-1) and δH 5.89 (dd, J = 10, 5.1 Hz, H-2); and the remaining two are doublets at δH 5.83 and 5.80 attributed to methylenedioxy group with a geminal coupling. The typical AB pattern of the benzylic H-6 protons at δH 4-4.5 that is characteristic for all C-6 unsubstituted alkaloids of the crinine series was absent in the 1 H NMR spectrum of 1 , and instead it showed only one singlet at δH 5.05 for H-6α. The absence of the typical AB pattern together with the pronounced deshielding effect on H-6α as well as C-6 in 13C-NMR spectrum suggested oxylation of C-6. Further investigation of 1 H NMR spectrum of 1, indicated that the large coupling constant measured from the splitting of H-4α signal δH 3.79 (dd, J = 13.6 Hz) and H-4α, led to trans-diaxial configuration  . A multiplet at δH 3.74 was assigned to H-3, whereas the two (ddd) at δH 1.8 and 1.5 were assigned to H-4α and H-4β, respectively. The large coupling constant between H-3 and H-4α (13.6 Hz) as well as the NOE correlation between H-3 and H-4α were indicative to the cis relationship between the C-3 pseudoequatorial substituent and the 5, 10b-ethano bridge. It was clear from 13 C spectral chemical shift of the two methoxyl groups δc 56.9 (6-OCH 3 ), 56.7(3-OCH 3 ), that they are aliphatic, this deduction supported by 1 H chemical shifts δH 3.32 (5, 6-OCH 3 ) and 3.25 (5, 3-OCH 3 ). The former was further deshielded due to the nitrogen atom of ring B. The observed NOESY and HMBC correlations between the 6-OMe group and H-6α, and C-6β, respectively, proved the presence of the methoxyl group at C-6. The coupling pattern of protons of the ethano-bridge that was represented by only three (dd) at δH 3.65, 2.74 and 2.02, together with the observed deshielding of H-11endo as well as C-11 in 13 C-NMR in comparison with the related C-11 unsubstituted alkaloids indicated the oxylation at this position. The NOE effect between H-10 and H-11endo was consistent with a hydroxyl substituent at the exo-position. The 13 C-NMR spectrum of compound 1 showed a similarity to its related alkaloid crinamine but with a pronounced deshielding of C-6 to be at δc 87.3 and the appearance of an additional aliphatic signal at δc 56.9 for C 6 -OMe group. All these findings together with the coincidence of its physical and spectral data , indicated that compound 1 is 6-methoxy crinamine, which was previously isolated from C. zeylanicum  and this is the first time to be isolated from Crinum augustum Rox.
Negative and positive modes HR-ESI-MS of 2 revealed molecular ion peak at m/z 300.110 [M-H] - and m/z 302.13 [M+ H]+ respectively, showed it to have the molecular formula C 17 H 19 NO 4 . UV spectral data of 2 , indicated to the presence of three maxima absorption (λmax 292, 241 and 208), which indicated the presence of a methylenedioxy-substituted benzene ring  , indicated the presence of crinine nucleus. 13 C NMR spectra ( 1 H decoupled) of 2 , showed seventeen resonances [Table 3]. 1 H NMR spectral data of 2 showed two singlets for the two para aromatic protons of ring A at δH 6.71 and 6.39 that were assigned to H10 and H-7, respectively. H-10 could be distinguished from H-7 through the observed NOESY correlations of H-10 with H-1 and H-7 with 2H-6. Another broad singlet was observed at δH 6.16 for two olefinic protons, their multiplicities were in agreement with the cis-relationship between the C-3 substituent and the 5,10b-ethano-bridge. This olefinic part of the spectrum showed a close resemblance with that of alkaloids of the (+)-crinane-skeleton  . The methylenedioxy group was assigned by the presence of the characteristic doublets δH 5.81 and 5.80 (J =1.4 Hz, H2-15). Two doublets characteristic for the typical AB pattern of the benzylic H-6 protons were observed at δH 4.2 and 3.58 assigned to H-6β and H-6α, respectively, with a large coupling constant (J = 17 Hz). 2D NOE correlations between H-4a and the low field signal at δH 4.2 and between H-12 endo and the up field signal δH 3.58 confirmed the previous assignment. The spectrum showed a doublet of doublet δH 3.88 was assigned for H-3. The large coupling between H-3 and H-4α (J = 9 Hz) as well as the NOE correlation between H-3 and H-4α were also indicative of the cis-relationship between the C-3 pseudoequatorial substituent and the 5,10b-ethanobridge  . Another double doublet δH 3.14 was assigned to H-4α and two (ddd) at δH 2.04 and 1.98 were assigned to H-4α and H-4β, respectively. A singlet δH 3.32 was indicative for the C-3 methoxy group. The coupling pattern of protons of the 5,10b-ethano bridge that were represented by the three doublets at δH 3.92, 3.30 and 3.25 together with the pronounced deshielding of H-11 endo as well as C-11 in 13 C NMR in relation to alkaloids with no bridge substituents were indicative to substitution at C-11. In addition, the NOE effect between H-10 and H-11 endo was consistent with a hydroxyl substituent at the exo-position. The carbon signal of C-9 was assigned at lower field than C-8 because of its three bond connectivities with H-7, in addition to, the quaternary carbons C-6a and C-10a were ascribed by means of their HMBC correlations with H-10 and H-7, respectively. The aliphatic region of the spectrum was characterized by one singlet for the quaternary carbon C-10b, three singlets for the methine carbons C-3, C-4a and C-11, three singlets for the methylene carbons C-4, C-6 and C-12, and one singlet for the methoxyl group at C-3. From the previous data, the compound 2 is crinamine, which was previously isolated from Crinum augustum Rox.  .
Positive and negative modes HR-ESI-MS of 3 revealed the molecular ion peak at m/z 286.154 [M+H] + and 284.0 [M-H] - respectively and showed it has the molecular formula C 17 H 19 NO 3 . UV spectrum of 3 , exhibited three maxima absorption (λmax 292, 241 and 208 nm) which indicated the presence of a methylenedioxy-substituted benzene ring and is coincided with the crinine nucleus  . The 13 C NMR spectrum of 3 showed 17 signals. The aliphatic region of the spectrum was characterized by one singlet for the quaternary carbon, two singlets for the methine carbons, four singlets for the methylene carbons, and one singlet for the methoxy group. 1 H NMR spectrum of 3 showed two singlets for the two-para aromatic protons H-10 and H-7 of ring A at δH 6.77 and 6.47, respectively. The observed NOE correlations is between H-10 and H-1; H-7 and H-6. The olefinic part of the spectrum showed a close resemblance with that of alkaloids of the (-)-crinine-skeleton  . The two olefinic protons appeared at δH 6.38 (d, J = 10 Hz, H-1) and δH 6.04 (ddd, J = 10, 5.1, 1.1 Hz, H-2) as an AB pattern of an ABX system partially overlapped by the methylenedioxy signals that appeared as doublet at dH 5.85 and 5.87 (J = 1.4 Hz) due to geminal coupling. The olefinic proton H-2 was further splitted by H-4B. Two doublets characteristic for the typical AB pattern of the benzylic H-6 protons were observed at δH 4.63 ppm and 4.06 ppm assigned to H-6α and H-6b, respectively. H-6α was shifted downfield due to the nitrogen lone pair  . The 2D NOE correlations between H-4α and the low field signal at δH 4.63 ppm and between H-12 endo and the up field signal at dH 4.06 distinguished the two protons and confirmed the previous assignment. Their large coupling (J = 16.3 Hz) is due to their trans-diaxial configuration  . The multiplet at δH 3.77 was assigned to H-3, while the singlet at δH 3.27 was assigned to the methoxy group at C-3. Its stereochemistry could be determined at the pseudoaxial configuration due to the large coupling (J = 5.1 Hz) between H-3 and H-2, the small coupling (J = 3.9 Hz) between H-3 and H-4β, the small coupling (J = 1.9 Hz) between H-3 and H-4α and the absence of allylic coupling between the vinylic H-1 and the allylic H-3. A doublet of doublet at δH 3.69 was assigned to H-4α which coupled with H-4α (J = 3.9 Hz) and H-48 (J = 13.4 Hz). Both signals of H-4α and H-4β were further splitted into (ddd) at δH 2.4 and 1.62, respectively, due to their mutual coupling (J = 13.4 Hz). The protons of the ethano-bridge were represented by the (ddd) at δH 2.2, 2.07, 3.12 and 3.84 excluding substitution at both C-11 and C-12. The lower field signal at δH 3.84 was attributed to H-12 exo not to H-12 endo because of its co-planarity with the nitrogen lone pair and the NOE correlation with H-4b  . The assignment of 2H4, H-4a, 2H-11 and 2H-12 were confirmed by 1H-1H COSY and NOESY experiments. The previous assignments pointed at the alkaloid buphanisine, which was isolated from Crinum augustum  .
Negative mode HR-ESI-MS of 4 revealed molecular ion peak at m/z 264.00 [M-H] - and indicates a molecular mass of 265 which corresponds to the molecular formula C 16 H 11 NO 3 . UV spectrum of 4 exhibited three maxima absorption (λmax 259, 226 and 208 nm) characteristic for alkaloids of the lycorine type  . 1HNMR spectrum showed six singlets in addition to two other triplet-like signals. The absence of the AB system of the benzylic H-7 protons that was replaced by the most downfield singlet at δH 9.26 was characteristic to the olefinic proton H-7 which is more deshielded by the nitrogen in these quaternary alkaloids of the lycorine type  . Four aromatic singlets at δH 7.93, 7.59, 7.53 and 7.25 ppm, evidently indicated the presence of two sets of tetra-substituted benzene ring system that were assigned to H-11 and H-8 of ring A as well as H-1 and H-3 of ring C, respectively. The other singlet at δH 6.28 assigned to methylenedioxy group attached to ring A as a common feature among Amaryllidaceae alkaloids. Protons of ring D wre represented by the two triplet-like signals at δH 5.13 and 3.66 with a vicinal coupling constant of 6.6 assigned to 2H-5 and H 2 -4, respectively; the former suffering more deshielded due to the neighboring nitrogen atom. The physical, chromatographic properties and spectral data of compound 4 are consistent with the data reported in literature  , so that it was identified as the alkaloid ungeremine, which was previously isolated from Crinum augustum Rox.  .
ESI-MS spectrum m/z 255.00 [M-HT] - (negative mode) of 6 , showed to have the molecular formula C16H32O2. Investigation of 1 H and spectral data led to classification of compound 6 as an aliphatic compound with a single double bond. 1 H NMR spectrum of 6 showed a triplet at δH 0.78 (J = 7 Hz) that was assigned to CH 3 -14 representing a vicinal coupling with an adjacent methylene group. The crowded multiplets at δH 1.11-1.20 were suggestive of a long carbon chain of methylene groups. A triplet at δH 2.18 (J = 7.5) that was assigned to CH 2 -2 indicated the attachment of this methylene group to a carbonyl function while the magnitude of the coupling constant were indicative to a vicinal coupling with an adjacent methylene group that was assigned to CH 2 -3 and resonated as a multiplet at δH 1.51. The quartet at δH 4 (J = 7), that was assigned to CH 2 -1\ , was characteristic for a direct attachment to an oxygen atom and a vicinal coupling with an adjacent methyl group CH 3 -2\ , the later was overlapped with the crowded region at δH 1.11-1.20. The previously suggested coupling patterns told that the 1 H NMR spectrum of 6 is typical for a saturated fatty acid ethyl ester, that together with the obtained molecular formula, it could be identified as myristic acid ethyl ester. Myristic acid was previously isolated from C. americanum L. and C. augustum Rox.  , while this is the first time for isolation of myristic acid ethyl ester from the genus Crinum.
The HR-ESI-MS (negative mode) spectrum of compound 7 showed it to have the molecular formula C l8 H 36 O 2 . 1H NMR spectrum of 7 showed a large similarity to that of 6 indicating that they belong to the same homologous series. The triplet at δH 0.78 was assigned to CH3-16, while the triplet at δH 2.2 (J = 7.3 Hz) that was assigned to CH 2 -2 indicated the attachment of this methylene group to the carbonyl group of the carboxyl function and the magnitude of the coupling constant was typical to a vicinal coupling with the adjacent CH2- 3 group that resonated as a multiplet at δH 1.9-2.0. The ethyl group of the fatty acid ethyl ester could be simply detected through the quartet at δH 4 (J = 7.3) that was assigned to CH 2 -1\ adjacent to the carboxyl function and was coupled with CH 3 -2\ that resonated as a triplet in the crowded region of the other methylene groups of the fatty acid chain at δH 1.16-1.25. The crucial step of determining the chain length of the fatty acid could be attained through the HR- that ESI-MS indicating compound 7 is palmitic acid ethyl ester. Palmitic acid as well as its methyl ester were previously isolated from C. bulbispermum Milne. and C. augustum Rox. 26 , while this is the first time for isolation of palmitic acid ethyl ester from the genus Crinum.
Finally, compounds 1 , 6, 7 and 8 are first report in Crinum augustum. Compound 1 is isolated from C. zeylanicum  and this is the first time to be isolated from Crinum augustum Rox. Myristic acid was previously isolated from C. americanum L. and C. augustum Rox.  , while this is the first time for isolation of myristic acid ethyl ester from the genus Crinum.
The effect of compounds 1 , 2 and 3 on settlement and variability of cyprids [Table 1] and [Figure 2], with EC50; 1.8, 1.2 and 0.75 μg/mL respectively. CuSO 4 (EC 50 = 0.23 μg/mL) was used as a positive control. Compounds 1 and 2 significantly affected settlement at 1 μg/mL and completely inhibited at 3 μg/mL. The other compounds (4, 6, 7, 8) showed no activity up to10 mg/mL. Compounds 1-3 have the same nucleus, thus, their mechanism of action are probably similar. This suggestion explains the similarity of their safety which coincided with CuSO4 safety.
Many of the amaryllidaceous alkaloids are biosynthetically created by a phenol activated oxidative coupling reaction. In the laboratory a large number of oxidising agents have been successful in emulating nature  .
6-0Me-Crinamine (1): (39 mg, 99% purity); yellowish oil; R f 0.48, silica gel 60 F 254 (Pet. ether-EtOAc 1:1); failed to respond to many trials of crystallization; [α] D 20 = + 20.2° MeOH; c. 0.0025 g/ml). UV λmax nm: 292 (ε = 96652), 241 (ε = 79771), 205 (ε = 67855); 1 HNMR (400 MHz, CDCl 3 ): δ 6.9 (1H, s, H-10), 6.71 (1H, s, H-7), 6.47 (1H, d, J = 10.5 Hz, H-1), 5.89 (1H, ddd, J = 10, 5.1 Hz, H-2), 5.83-5.80 (2H, 2d, J = 1.2 Hz, OCH2O), 5.05 (1H, s, H-6α), 3.79 (1H, dd, J = 13.6, 4.4 Hz, H-4a), 3.74 (1H, m, H-3), 3.65 (1H, dd, J = 9, 4.6 Hz, H-11 endo), 3.32 (3H, s, 6-OCH 3 ), 3.25 (3H, s, 3- OCH 3 ), 2.74 (1H, dd, J = 13.2, 9 Hz, H-12 endo), 2.02 (1H, dd, J = 13.2, 4.6 Hz, H-12 exo), 1.8 (1H, ddd, J = 13.6, 13.6, 4 Hz, H-4α), 1.5 (1H, ddd, J = 13.6, 4.4, 4.1 Hz, H-4b), 13 C-NMR (75 MHz, CDCl 3 ): 147.7 (s, C-9), 146.4 (s, C-8), 139 (d, C-1), 137.6 (s, C-10a), 127.6 (s, C-6a), 126.3 (d, C-2), 109.9 (d, C-7), 102.9 (d, C-10), 102.7 (t, OCH 2 O), 87.3 (t, C-6), 77.6 (d, C-11), 72.6 (d, C-3), 62.4 (d, C-4a), 57.1 (t, C-12), 56.9 (q, 6-OCH 3 ), 56.7 (q, 3-OCH 3 ), 45.3 (s, C-10b), δ 28.3 (t, C-4); HRESI- MS [positive mode] m/z 332.14 [M+H] + (calculated for C 18 H 21 NO 5 , 331.14203).
Crinamine (2); (60 mg, 100% purity); colorless needles; R1 0.41, silica gel 60 F 254 (Pet. ether-EtOAc 1:1); mp.200 C ;[α] D 20 = + 356.8˚ (Me0H; c. 0.0025 g/ml). UV λmax nm: 292 (ε = 87892), 241 (ε = 72541), 208 (ε = 62608). 1 H-NMR (400 MHz, CDCl 3 ): δ 6.71 (1H, s, H-10), 6.39 (1H, s, H-7), 6.16 (2H, s, H-1 and H-2), 5.81 5.80 (2H, 2d, J = 1.3 Hz, OCH 2 O), 4.20 (1H, J = 17 Hz, H-6β), 3.92 (1H, dd, J = 6, 4 Hz, H-11 endo), 3.58 (1H, d, J = 17 Hz, H-6α), 3.32 (3H, s, 3-OCH 3 ), 3.30 (1H, dd, J = 13.9, 6 Hz, H-12 endo), 3.25 (1H, dd, J = 13.9, 4 Hz, H-12 exo), 3.14 (1H, dd, J = 13, 4.6 Hz, H-4a), 2.04 (1H, ddd, J = 13, 11.7, 9 Hz, H-4α), 1.98 (1H, ddd, J = 11.7, 6, 4.6 Hz, H-4β). 13C-NMR (75 MHz, CDCl3 ): δ 146.5 (s, C-9), 146.2 (s, C-8), 135.9 (d, C-1), 135.5 (s, C-10a), 126.7 (s, C-6a), 123.7 (d, C-2), 106.8 (d, C-7), 103.2 (d, C-10), 100.8 (t, OCH 2 O), 80 (d, C-11), 76.1 (d, C-3), 66.2 (d, C-4a), 63.6 (t, C-6), 61.2 (t, C-12), 55.7 (q, 3-OCH 3 ), 50.3 (s, C-10b), 30.2 (t, C-4). HR-ESTMS [positive mode] m/z 302.13 [M+H] + and [negative mode] m/z 300.11 [M H] - (calculated for C 17 H 19 NO 4 , 301.13147).
Buphanisine (3); (34.5 mg, 100% purity); yellowish oil; R f 0.49, silica gel 60 F 254 (Pet. ether-EtOAc 1:1); failed to respond to many trials of crystallization; [α] D 21 = -39.2° (Me0H; c 0.0025 g/ml). UV λmax nm: 292 (ε = 83320), 241 (ε = 68685), 208 (ε = 59280). 1 H-NMR (400 MHz, CDCl 3 ): δ 6.77 (1H, s, H-10), 6.47 (1H, s, H-7), 6.38 (1H, J = 10 Hz, H-1), 6.04 (1H, ddd, J= 10, 5.1, 1.1 Hz, H-2), 5.85-5.87 (2H, 2d, J = 1.4 Hz, OCH 2 O), 4.63 (1H, d, J = 16.3 Hz, H-6α), 4.06 (1H, d, J = 16.3 Hz, H-6β), 3.84 (1H, ddd, J = 13.4, 11, 4 Hz, H-12 exo), 3.77 (1H, m, H-3), 3.69 (1H, dd, J = 13.4, 3.9 Hz, H- 4a), 3.27 (3H, s, 3-OCH 3 ), 3.12 (1H, ddd, J = 13.4, 9, 6.6 Hz, H-12 endo), 2.4 (1H, ddd, J = 13.4, 3.9, 1.9 Hz, H-4α), 2.2 (1H, ddd, J = 12.7, 9, 4 Hz, H-11 endo), ), 2.07 (1H, ddd, J = 12.7, 11, 6.6 Hz, H-11 exo), 1.62 (1H, ddd, J = 13.4, 13.4, 3.9 Hz, H-4β). 13 C-NMR (75 MHz, CDCl 3 ): δ 147.8 (s, C-9), 147.3 (s, C-8), 135.9 (s, C-10a), 130.1 (d, C-1), 127 (d, C-2), 121 (s, C-6a), 107.5 (d, C-7), 103.7 (d, C-10), 101.7 (t, OCH2O), 71.6 (d, C-3), 64.3 (d, C-4a), 60.3 (t, C-6), 57.2 (q, 3-OCH3), 52.8 (t, C-12), 45.3 (s, C-10b), 42.1 (t, C-11), 26.7 (t, C-4). HR-ESI-MS [positive mode] m/z 286.154 [M+H] + and [negative mode] m/z 284 [M H] - (calculated for C 17 H 19 NO 3 , 285.13657).
Ungeremine (4); (42 mg, 99% purity); yellow crystals; R f 0.39, silica gel 60 F 254 (CHCl 3 -Me0H 8:2); mp. 256257 ˚C ;UV λmax nm: 259 (ε = 68635), 226 (ε = 59890), 208 (ε = 55120). 1 H-NMR (400 MHz, CD 3 OD): β 9.26 (1H, s, H-7), 7.93 (1H, s, H-11), 7.59 (1H, s, H-8), 7.53 (1H, s, H-1), 7.25 (1H, s, H-3), 6.28 (2H, s, OCH2 O), 5.13 (2H, t-like, J = 6.6 Hz, 2H-5), 3.66 (2H, t-like, J = 6.6 Hz, 2H-4), HR-ESI-MS [negative mode] m/z 264 [M-H] - (calculated for C 16 H 11 NO 3 , 265.07393).
Myristic acid ethyl ester (6); (199 mg, 100% purity); yellowish oil; R f 0.46, silica gel 60 F 254 (pet. etherCHCl 3 8:2); 1 H-NMR (400 MHz, CDCI 3 ): δ 4.00 (2H, J = 7 Hz, I-CH 2 ), 218 (2H, t, J = 7.5 Hz, 2-CH 2 ), 1.51 (2H, p, J = 7.3 Hz, 3-CH 2 ), 1.11-1.20 (m, 2'-CH 3 and other CH 2 protons), 0.78 (3H, J = 7 Hz, 14-CH 3 ). HR-ESI-MS [negative mode] m/z 255 [M-H] - (calculated for C 16 H 32 O 2 , 256.24036).
Palmitic acid ethyl ester (7); (12.7 mg, 99% purity); yellowish oil; R f 0.69, silica gel 60 F 254 (pet. etherCHCI 3 8:2); 1 H-NMR (400 MHz, CDCl 3 ): δ 4.00 (2H, q, J = 7.3 Hz, 1-CH 2 ), 2.2 (2H, t, J = 7.3 Hz, 2-CH 2 ), 1.9-2 (2H, m, 3-CH 2 ), 1.16-1.25 (m, 2'-CH 3 and other CH2 protons), 0.78 (3H, t, J = 7.3Hz, 16-CH 3 ). HR-ESI-MS [negative mode] m/z 283.26 [M-H] - (calculated for C 18 H 36 O2 , 284.27168).
Ursolic acid (8); (26.5 mg, 99% purity); yellowish white powder; R f 0.49, silica gel 60 F 254 (pet. ether-EtOAc 9:1); mp.260-262°C; 1 H-NMR (400 MHz, CDCl 3 ): δ 5.12 (1H, br.s, H-12), 4.4 (1H, d, J = 7, H-3α), 2.18 (1H, d, J = 11.5, H-18β), 0.9-1.9 (m, CH 2 groups), 0.76-0.82 (m, CH 3 groups). HR-ESI-MS [negative mode] m/z 455 [M-H] - (calculated for C30 H48 O 3 , 456.36054).
β-sitosterol-O-glucoside (9); (26 mg, 100% purity); white amorphous powder; R f 0.23, silica gel 60 F 254 (CHCl 3 -Me0H 9:1); mp.276-277°C.
| Acknowledgement|| |
The authors are gratefully thanks professor A. rayed (Plant Taxonomy Faculty of Science Assiut University, Egypt) for his effort in the identification of plant material. Thanks go to Mr. Hirroshi Matsuura (Faculty of environmental earth science, Hokkaido University, Sapporo, 060-0810, Japan), for his help in measuring the different Mass spectra. [Table 2]
| References|| |
|1.||Benson, L., Handbook of Plant Classification, (Oxford and I.B.H publishing Co., New Delhi, Bombay, 1970) 793. |
|2.||W. Meerow, D. A. Snijman, Families and Genera of Vascular Plants, (Springer-Verlag, Berlin, 1998, Vol. III) 83. |
|3.||W. C. Wildman, The Alkaloids: Chemistry and Physiology, Vol. VI, (R.H.F. Manske, Academic Press, New York, London 1960). |
|4.||M. Leven, D. Van den Berghe, A.J. Vlietinck, J. Totte, R. Dommisse, E. and Esmans, F. Alderweireldt. Plant antiviralagents. III. Isolation of alkaloids from Clivia miniata Regel (Amaryllidaceae). J Nat Prod. 45: 564-73 (1982). |
|5.||J. R. Lewis. Amaryllidaceae and Sceletium alkaloids. Nat.Prod. Rep. 13: 171-6 (1996). |
|6.||Kaduszkiewicz, T. Zimmermann, H. P. Beck-Bornholdt and H. Van den Busschi, Cholinestrase inhibitors for patients with Alzheimer's disease: systematic review of randomized clinical trials. BMJ. 331: 321-7(2005). |
|7.||N. T. N. Tram, T. Titoren-Kava, V. Bankova, N. Handjieva and S. S. Popova. Crinum L. Amaryllidaceae, Fitoterapia, 73: 183- 208, (2002). |
|8.||C. W. Fennel and J. Vanstaden. Crinum species in traditional and modern medicine. J Ethinopharmacol. 78: 15-26. |
|9.||N. Fuestani, H. Hirota, T. Okino, Y. Tomono, and E. Yoshimura. Antifouling activity of isocyanoterpenoids and related compounds isolated from a marine sponge and nudbrancchs. J Nat Toxins. 5: 349-59 (1996). |
|10.||Fuestani. Biofouling and antifouling. Nat. Prod. Rep. 21: 94- 104 (2004). |
|11.||Y. Kitano, A. Yokoyama, Y. Nogota, K. Shinshima, E. Yoshimura, K. Chiba, M. Tada, I. Sakaguch I., Synthesis and anti-brancle activity of novel 3-isocyanotheonellin analogues.Biofouling, 19: 87-192 (2003). |
|12.||D. Rittschof, A. S. Clare, D. J. Gerhart, S. Avelin, and J. Bonaventura. Brancle In-Vitro assays for biologically active substances. Biofouling 115-122 (1992). |
|13.||S. Ghosal, K.S. Saini and S. Razdan. Crinum alkaloids: their chemistry and biology. Phytochemistry. 24: 2141-2156 (1985). |
|14.||F. Viladomat, J. Bastida, C. Codina, W. E. Campbell, and S. Mathee Alkaloids from Brunsvigia josephinζ. Phytochemistry. 35: 809-12 (1994). |
|15.||Y. Tsuda, N. Kashiwaba, and V. Kumar. The alkaloidal constituents of goda-manel (Crinum zeylanicum L.) A Sri Lankan folk medicine. Chem Pharm Bull. 32: 3023-7 (1984). |
|16.||F. Viladomat, G. R. Almanza, C. Codina, J. Bastida, W. E. Campbell, and S. Mathee. Alkaloids from Brunsvigia orientalis. Phytochemistry. 43: 1379-84 (1996). |
|17.||A. A. Ali, H. Kating, and A.W. Frahm, Four 6-hydroxylated alkaloids in the crinine series from Crinum augustum Rox. Phytochemistry. 20:1731-3 (1981). |
|18.||F. Viladmat, C.Cadina, J. Bastida, S. mathee and W.E. Campbell. Further alkaloids from Brunsvigia josephinae. Phytochemistry 40: 961-965 (1995). |
|19.||J. J. Nair, A. K Machocho,W. E. Campbell, R. Brun, F. Viladomat, C. Codina and J. Bastida. Alkaloids from Crinum macowanii. Phytochemistry. 54: 945-950 (2000). |
|20.||A.A. Ali, H. Kating, A.W. Frahm, A.M. El-Moghazi, and M.A Ramadan. Four 6-hydroxylated alkaloids in the crinine series from Crinum augustum Rox. Phytochemistry. 20:1121-3 (1981). |
|21.||K. Rhee, N. Apples, B. Hofte, B. Karabatak, C. Erkelens, L. M. Stark, L. A. Flippin, R. and Verpoorte, R. Isolation of acetylcholinestrase inhibitor ungeremine from Nerine bowdenii by preparative HPLC coupled with on-line to a flow assay system. Bio Pharm Bull. 27: 1804-9 (2004). |
|22.||M. A Ramadan. Investigation of petroleum ether extract of the bulbs of Crinum augustum Rox. Bull Pharm Sci Assiut Univ. 21: 97-102 (1998). |
|23.||M. A. Abd El-Hafiz, and A.M. Mesbah, Investigation of mucilages and fatty acids of Crinum augustum Rox. and Crinum americanum L. Egypt J Pharm Sci. 25: 171-8 (1984). |
|24.||M. A. Abd El-Hafiz. Aliphatic hydroxyketones from Crinum augustum. Phytochemistry 30: 3127-3129, (1991). |
|25.||J. R. Lewis. Amaryllidaceae and Sceletium alkaloids. Nat Prod Rep. 17: 57-84 (2000). |
[Figure 1], [Figure 2]
[Table 1], [Table 3], [Table 2]