Two New Sesquiterpene Coumarins, Ferusinol and Samarcandin Diastereomer, from Ferula sinaica

Authors

Abstract

Re-investigation of the methylene chloride extract of the roots of Ferula sinaica gave ferusinol, a new sesquiterpene coumarin with a rare carbon skeleton, and samarcandin diastereomer. The structures elucidation were determined by MS, 1H- 13C-1D and 2D NMR spectral data.

Keywords


Acute and Subchronic Toxicity?of Teucrium polium Total Extract in Rats
Iranian Journal of Pharmaceutical Research (2007), 6 (3): 217-221
Received: July 2006
Accepted: December 2006

Copyright ? 2007 by School of Pharmacy
Shaheed Beheshti University of Medical Sciences and Health Services

Original Article

Two New Sesquiterpene Coumarins, Ferusinol and Samarcandin

Diastereomer, from Ferula sinaica

 

Ashraf A. El-Bassuonya, Ahmed A. Goharb and Amal M. Kabbashc*

aBasic Sciences Department, Industrial Education College, Beni Suef University, Egypt. bPharmacognosy Department, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt. cPharmacognosy Department, Faculty of Pharmacy, Tanta University, Tanta, Egypt.

 

Abstract

Re-investigation of the methylene chloride extract of the roots of Ferula sinaica gave ferusinol, a new sesquiterpene coumarin with a rare carbon skeleton, and samarcandin diastereomer. The structures elucidation were determined by MS, 1H- 13C-1D and 2D NMR spectral data.

 

Keywords: Ferula sinaica; Apiaceae; Sesquiterpene coumarins.

Introduction

The genus Ferula belongs to the family Apiaceae with some 130 species distributed throughout the Mediterranean area and Central Asia (1). Several species of Ferula have been used in folk medicine. Thus, F. communis L. and its subspecies and varieties have been used as agents against hysteria and to treat dysentery (2), F. jaeschkeana Vatke has been applied as a herbal contraceptive (3) and F. tingitana L. has proved to be a good source of ammoniac, an oleo-gum resin used in medicine (4).

The widespread sesquiterpene compounds in this genus are characteristic daucanes, humulanes, himachalanes, germacranes, eudesmanes, and guaiananes (5). The study of the chemical constituents of this genus has developed rapidly over the last twenty years due to more efficient methods of purification and the availability of sophisticated techniques for structure elucidation and thus several types of sesquiterpene coumarin compounds have been isolated (6-10).

Previous works on F. sinaica led to isolation of sesquiterpene coumarins, daucanes, bornanes and monoterpenes (6, 11-12). In continuation of our interest in the chemical constituents of the Egyptian medicinal plants, we re-investigated the roots of F. sinaica L. and found a new sesquiterpene coumarin with a rare carbon skeleton, named ferusinol, and a diastereomer samarcandin (Figure 1).

 

Experimental

General

1H-NMR (500 MHz, CDCl3). 13C-NMR (125 MHz, CDCl3) and 2D spectra were recorded on a JEOL 500 MHz, Lambda spectrometer, TLC: precoated silica gel type 60 (Merck). HPLC was performed in the reversed phase mode on Knauer pump 64 and different refractometer (column: RP-8, 250?25 mm, flow=17 ml/min, elution with MeOH-H2O, mixtures, refractive index).

 

Plant material

The fresh roots of F. sinaica L. (Apiaceae) (3 kg) were collected from Sinai, Egypt, March 2002. The fresh roots were cutted into slices, air-dried and powdered. A voucher specimen (A. A. 111) is deposited at the Department of Botany, Faculty of Science, Beni Suef, Cairo University.

 

Extraction and separation

The air-dried plant materials (3 kg) were ground and extracted at room temperature with CH2Cl2-MeOH (1:1). The extracts were concentrated in vacuum to leave 200 g. residue. This residue was fractionated on flash column, silica gel, using n-hexane, CH2Cl2, increased polarity. The fractions eluted with 75% CH2Cl2 was refractionated by CC (6 ?120 cm) on silica gel eluting with n-hexane followed by a gradient of n-hexane-CH2Cl2 up to 100% CH2Cl2 then CH2Cl2-MeOH up to 15 % MeOH. The 100% CH2Cl2 fraction was further purified by CC (2?40 cm), on Sephadex LH-20 eluted with n-hexane-CH2Cl2-MeOH (6: 4: 1) to give a mixture of 1 (9 mg) and 2 (5 mg). The mixture was purified by HPLC (MeOH-H2O, 65:35, Rt= 5.6 and 6.0 min).

 

Bioassay

The antibacterial activity of ferusinol and samarcandin diasteriomer were determined against Gram-negative strains (Serratia sp., Pseudomonas sp., Escherichia coli) and Gram-positive bacteria (Bacillus cereus, Staphylococcus aureus), obtained from culture collection of Bacteriological Laboratory, Department of Botany, Faculty of Science, El-Minia University, Egypt, using Whatman filter paper No. 1, 1 cm diameter, disc diffusion assay methods. Five replicates were performed for the compounds at concentrations 200 ?g/ml and 400 ?g/ml. Discs were soaked in the test compound for 30 sec, dried and then laid on the surface of nutrient agar medium inoculated with the test bacterium. The plates were incubated at 30 ?C for 48 h. Ampicillin (purchased from ADWIC Company, Egypt) and amoxillin (purchased from ADCO Company, Egypt) were used as a reference compounds.

 

Ferusinol 1

1H-NMR (500 MHz, CDCl3): δ 6.26 (1H, d, J= 9 Hz, H-3), 7.63 (1H, d, J= 9 Hz, H-4), 7.63 (1H, d, J= 9 Hz, H-5), 6.83 (1H, dd, J= 2.5, 9 Hz, H-6), 6.81 (1H, d, J= 2.5 Hz, H-8), 1.10 (1H, m, H-1`a), 0.95 (1H, m, H-1`b), 1.15 (1H, m, H-2`a), 1.95 (1H, m, H-2`b), 2.11 (1H, ddd, J= 3.5, 6, 6 Hz, H-3`), 1.41 (1H, ddd, J= 3.5, 13, 13 Hz, H-5`), 1.90 (1H, ddd, J= 4, 13, 13 Hz, H-6`a), 1.49 (1H, ddd, J= 4, 13, 13 Hz, H-6`b), 3.39 (1H, ddd, J= 4, 13, 13 Hz, H-7`), 1.96 (1H, dd, J= 2.5, 13 Hz, H-8`), 2.23 (1H, ddd, J= 2.5, 9, 9 Hz, H-9`), 4.05 (1H, dd, J= 7, 9 Hz, H-11`a), 3.97 (1H, dd, J= 7, 9 Hz, H-11`b), 1.19 (3H, d, J= 7 Hz, H-12`), 1.65 (3H, s, H-13`), 5.05 (1H, br s, H-14`a), 4.91 (1H, br s, H-14`b), 0.93 (3H, s, H-15`); CIMS (M + H)+ m/z 383 (C24H30O4) (30 %), 365 (M + H ? H2O)+ (60 %); IR 3345 (OH), 2963, 1726 cm-1 (C=O).

 

Samarcandin diastereomer 2

1H-NMR (500 MHz, CDCl3): δ 6.28 (1H, d, J 9.55 Hz, H-3), 7.63 (1H, d, J 9.55 Hz, H-4), 7.37 (1H, d, J 8.6 Hz, H-5), 7.19 (1H, dd, J 2.5, 8.6 Hz, H-6), 7.03 (1H, d, J 2.5 Hz, H-8), 1.45 (2H, m, H-1`), 2.05 (2H, m, H-2`), 3.42 (1H, dd, J 11, 2 Hz, H-3`), 1.57 (1H, dd, J 2, 8 Hz, H-5`), 1.15 (2H, m, H-6`), 1.35 (2H, m, H-7`), 2.00 (1H, dd, J 4.9, 10.2 Hz, H-9`), 4.66 (1H, d, J 6.65 Hz, H-11`a), 4.28 (1H, dd, J 6.65, 10.2 Hz, H-11`b), 1.39 (3H, s, H-12`), 1.20 (3H, s, H-13`), 1.04 (3H, s, H-14`), 1.02 (3H, s, H-15`); EIMS (M)+ m/z 400 (C24H32O3) (30 %), 382 (M ? H2O)+ (60 %); IR 3450 (OH), 1685 (α - pyrone), 1610, 1595, 1225, 1205 and 1150 cm-1.

Results and Discussion

The CH2Cl2-MeOH (1:1) extract of F. sinaica was partitioned by successive chromotographic separations to afford two new sesquiterpene coumarins, namely, ferusinol (1) and a diastereomer samarcandin (2). The structure of 1 was established by the analysis of its 1H-NMR and 13C-NMR spectra, (Table 1), which showed six quaternary carbons, ten tertiary carbons, five secondary carbons and three primary carbons. The 1H-NMR and 13C-NMR spectra suggested the presence of an umbelliferone moiety in the skeleton from the signals at δH 7.63 (δC 143.4, d, J= 9 Hz, H-4), δH 7.63 (δC 128.7, d, J= 9 Hz, H-5), δH 6.83 (δC 113.0, dd, J= 9, 2.5 Hz, H-6), δH 6.81 (δC 101.1, d, J=2.5 Hz, H-8) and δH 6.26 (δC 113.0, d, J= 9 Hz, H-3). The 1H-NMR data (in CDCl3, 500 MHz) of the sesquiterpene part exhibited a different pattern compared to the previously reported sesquiterpene coumarins in the genus Ferula. The protons and their sequences could be assigned by 1H-1H COSY as following: the two signals at δH 5.05 ( br s ) and δH 4.91 (br s ) were characteristic for exomethylene protons (H-14`a, H-14`b). The hydroxylated carbon (C-7`) appeared at δC 70.2 and H-7` at δH 3.39 (ddd, J= 4, 13, 13 Hz). The signal at δH 1.41 was characteristic for H-5` (ddd, J= 3.5, 13, 13 Hz), and the two protons H-11`a, H-11`b appeared as two double doublets at δH 4.05 (J= 7, 9 Hz), 3.97 (J=7, 9 Hz). The signal at δH 2.23 (ddd, J= 2.5, 9, 9 Hz) was assigned for H-9`. The three methyl protons H-12`, H-13` and H-15` appeared as one doublet and two singlets at δH 1.19, 1.65 and 0.93, respectively. The sesquiterpene moiety showed two olefinic carbons at δC 116.9 and 144.1, characteristic for C-14` and C-4`, respectively. The three methyl carbons C-12`, C-13` and C-15` appeared as δC 23.0, 24.6 and 17.9, respectively. The other carbon signals are reported in Table 1. The relative stereochemistry of 1 was deduced from NOE experiments; irradiation of H-7` enhanced H-5`, irradiation of H-15` showed an effect on H-11`, irradiation of H-3` enhanced H-5` and irradiation of H-5` enhanced H-7` and H-12`. Ferusinol had a molecular formula C24H30O4 indicated by CIMS, (M + H)+ at m/z 383 (30 %), (M + H ? H2O)+ at m/z 365 (60 %). The proposed biosynthetic route of ferusinol (1) is given in Scheme 1.

 

 

1H-NMR of compound 2, aided with 1H-1H COSY, showed two signals at δH 7.63 and 6.28 (each 1H, d, J= 9.5 Hz) assigned to H-4 and H-3, while the signals at δH 7.37 (1H, d, J= 8.6 Hz), 7.03 (1H, d, J= 2.5 Hz) and 7.19 (1H, dd, J= 2.5, 8.6 Hz) were assigned to H-5, H-8 and H-6 of the coumarin moiety, respectively. The methylene protons (H-11`) showed a different splitting than those reported in the literature, whereas, one of them appeared as double doublet signal at δH 4.28 (1H, dd, J= 6.65, 10.2 Hz), and the second appeared as a doublet signal at δH 4.66 (1H, d, J= 6.65 Hz). The two protons showed a 1H-1H COSY correlation with a double doublet signal at δH 2.00 (1H, dd, J= 4.9, 10.2 Hz, H-9`). An additional oxygenated proton (H-3`) was found as double doublet at δH 3.42 (1H, J= 11, 2 Hz). The methyl signals appeared at δH 1.39 (s), 1.20 (s), 1.04 (s), and 1.02 (s) were assigned to H-12`, H-13`, H-14` and H-15`, respectively. The 13C-NMR and DEPT experiments (Table 1) showed signals for 24 carbon signals, four quartets at δC 28.7, 28.0, 25.1 and 16.3 that were attributed to C-12`, C-13`, C-14` and C-15`, respectively, five triplet (four aliphatic methylenes at δC 45.0, 28.0, 20.7 and 38.3 being typical for C-1`, C-2`, C-6` and C-7`, respectively and one primary alcoholic carbon at δC 66.8 characteristic for C-11`, eight doublets at δC 77.8 (H-3`), 60.7 (H-9`), 55.3 (H-5`) five of which were due to the umbelliferone moiety at δC 112.9 (C-3), 143.8 (C-4), 129.3 (C-5), 113.4 (C-6) and 102.0 (C-8). The downfield signal at δC 162.796 was assigned to the carbonyl carbon of the coumarin moiety (C-2). Assignments of all protonated carbons were made by the analysis of the HMQC and HMBC. These data suggested that 2 was derived from sesquiterpene and umbelliferone components. The stereochemistry of 2 was deduced from NOE experiments; irradiation of H-3` resulted in enhancements of H-14` and H-5` signal, whereas irradiation of H-9` resulted in enhancement of Me-12`. Additionally, irradiation of H-15` showed a clear enhancement of Me-13`, indicating that α-orientation of H-3`, H-14` and β-orientation of H-9`, H-12`, H-13` and H-15`. EIMS of compound 2 showed a molecular ion peak at m/z 400 consistent with the molecular formula C24H32O3. The general feature of the data was in agreement with the samarcandin group (8-10). This is the first report of H-11` protons as one doublet and one double doublet, which suggest a new samarcandin diastreomer .

 

 

Compounds 1 and 2 were screend for their possible in vitro antibacterial activity against an assortment of two Gram-positive bacteria (B. cereus, S. aureus) and Gram-negative bacteria (Serratia sp., Pseudomonos sp., E. coli) using ampicillin and amoxillin as reference standards. The minimum inhibitory concentrations (MICs, μg/ml) were determined using standard agar dilution method (13). The MIC values are summarized in Table 2.

 

 

From the obtained data, it is clear that ferusinol possesses high activity against both Gram-positive strains, particularly B. cereus and Gram-negative ones. On the contrary, samarcandin diasteriomer showed higher activity against Gram-negative strains but it was inactive at tested concentrations against Gram-positive strains as shown in Table 2. Our results are in agreement with previous reports (14). Antibiotics such as ampicillin and amoxillin are bacterial cell wall synthesis inhibitors. Ferusinol may also inhibit cell wall synthesis in both Gram-positive and Gram-negative strains. On the other hand, samarcandin diastreomer may have similar mechanism of action, but on Gram-positive strains only. In other words, ferusinol and samarcandin diastereomer might act similar to penicillins against bacteria.

Acknowledgement

The authors thanks the Department of Botany, Faculty of Science, Beni Suef University for deposition of the voucher specimen of the plant.

References

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