Antibacterial coumarins isolated from Launaea resedifolia
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ANTIBACTERIAL COUMARINS ISOLATED FROM LAUNAEA RESEDIFOLIA
© Ashraf A. El-Bassuony1, N.M. Abdel-Hamid2
1Basic Science Department, Industrial Education College, Beni Suef University (Egypt) E-mail: ahnnbass@yahoo. com
2Biochemistry Department, Faculty of Pharmacy, El-Minia University (Egypt)
E-mail: nabilmohie@yahoo. com
From the aerial parts of Launaea resedifolia, four coumarin compounds were isolated from the methylene chloridemethanol (1: 1) extract, namely, cichoriin I, esculetin II, scopoletin III and isoscopoletin IV.
The structures of the compounds were elucidated by 1D, 2D-NMR and HR-CIMS analysis. These compounds showed high antibacterial activity against some Gram — positive bacteria as Bacillus cereus and Staphyllococcus aureus in minimum inhibitory concentrations of 200 and 400 |ig/mL. However, they showed no effect on tested Gram — negative bacteria as Serratia Sp, Pseudomonas Sp and Escherichia coli)
The genus Launaea (tribe Lactucaea, family Asteracea) comprises about 40 species. Many of its plants are used in folk medicine as bitter stomachic, for skin diseases, as antitumors and as insecticides. The genus Launaea presents possesses phytochemical features, such as terpenoids [1−5], phenolics [6−7], flavones  and coumarins [1, 9, 10]. Therefore, we investigated the chemical constituents of Launaea resedifolia collected from Algeria.
General experimental procedures. Perkin-Elmer 1000 FT-IR instrument was adopted for IR spectral analysis utilizing KBr pellets. The NMR spectra was recorded on Bruker AC 500 [500 MHz ('-H) and 125, MHz (13C)] spectrometer. Chemical shifts are recorded in 8 (ppm) using TMS as internal standard. EIMS were obtained at 70 ev using a VG-ZAB-E instruments. Column chromotography (CC) was performed using silica gel 60 (Merck, 0. 063−0.2 mm). TLC analysis was performed with silica gel (Merck, Kieselgel). Spots were visualized by UV (Xmax 259 and 360 mm). HPLC was performed in the reverse phase 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. Aerial parts of L. resedifolia, were collected in March 2002 from 25 km. North of Ouargla, Algeria, during flowering period. A voucher specimen was deposited at the herbarium of chemistry department, faculty of sciences, Constantine university, under the code number SR 101, Algeria.
Extraction and isolation procedures. The aerial parts of L. resedifolia (1 Kg) were dried, powdered and extracted with methylene chloride-methanol (1: 1) at room temperature. The solvent was distilled under reduced pressure furnishing a residue (10 g). The residue was submitted to flash column chromatography, being eluted with n-hexane, methylene chloride and methanol, increasing the degree of polarity. The extract was prefractionated by CC (6×120 cm) a silica gel eluting with n-hexane followed by a gradient of n-hexane-CH2Cl2 up to 100% CH2Cl2 and CH2Cl2-MeOH up to 15% MeOH. The fraction was further purified by CC (2×40 cm), a Sephadex LH-20 eluted with n-hexane-CH2Cl2-MeOH (6: 4:1) gave a complex mixture. The mixture was purified by HPLC (MeOH-H2O, 65: 35, Rt = 5.6 and 6,0 min).
* Автор, с которым следует вести переписку.
Bioassays. The antibacterial activity of compounds I-IV was determined against Gram-negative strains (Serratia sp., Pseudomonas sp., Escherichia coli) and Gram-positive bacteria (Bacillus cereus, Staphylococcus aureus), obtained from culture prepared in Department of Microbiology, Faculty of Pharmacy, El-Minia University, Egypt, using Whatman filter paper No. 1, 1 cm. Diameter, disc diffusion assay method. Five replicates were performed for the compounds with two concentrations (200 |ag/mL and 400 ^g/mL) of each compound were done. Discs were soaked in the test compound for 30 sec, evaporated, then overloaded on the surface of the nutrient agar media cultured with the tested bacterium. Ampicillin (purchased from ADWIC Comp., Egypt) and amoxillin (purchased from ADCO Comp., Egypt) were used as a reference compounds.
Results and discussion
Investigation of the CH2Cl2-MeOH (1: 1) extract of the aerial parts of Launaea resedifolia afforded four compounds. Compound I, colorless oil, CIMS showed a molecular ion peak [M+1]+ at m/z 341 in according with the molecular formula Ci5H16O9. The 13C-NMR spectrum of compound I displayed fifteen carbon signals. DEPT experiments indicated these signals as: one carbonyl carbon at 8C 160. 57 (s, C-2), one methylene carbon at 8C 60. 74 (t, C-6'-) — nine methine carbons at 8C 143. 59 (d, C-4), 112. 97 (d, C-3), 112. 65 (d, C-5), 103. 37 (d, C-8), 100. 99 (d, C-1'-), 77. 28(d, C-3'-), 75. 86 (d, C-2'-), 73. 17 (d, C-4'-) and 69. 81 (d, C-5'-) and four quaternary carbons at 8C 148. 81 (s, C-7),
147. 79 (s, C-8a), 144. 17 (s, C-4a) and 113. 45 (s, C-6). The: H-NMR spectrum showed characteristic signals of glucose moiety, whereas, the methylene protons H-6'-a and H-6'-b appeared as two double doublets at 8H 3. 93 (J = 12. 0, 3.0 Hz) and 3. 72 (J = 12. 0, 3.0 Hz). The anomeric proton H-1'- appeared downfield as doublet signal at 8H 4. 96 (J = 7.5 Hz), the other methin protons H-2'-, H-3'-, H-4'- and H-5'- appeared at 8H 3. 54 (dd, J = 7. 5, 8.5 Hz), 3. 50 (dd, J = 8. 5, 9.0 Hz), 3. 41(dd, J = 9. 0, 9.0 Hz) and 3. 51 (ddd, J = 3. 0, 5. 0, 9.0 Hz), respectively. The coumarin moiety exhibited characteristic signals as a doublet at 8H 7. 81 (H-4, J = 9.5 Hz), which correlated in 1H-1H COSY with doublet at 8H 6. 27 (H-3, J = 9.5 Hz). The two singlet signals appeared at 8H 7. 20 and 7. 03 was assigned for H-8 and H-5, respectively. All proton and carbon signals were assigned by 1H-1H and 1H-13C COSY. In the 1H-13C COSY, the signal at 8H 4. 96 (H-1'-) showed correlation with the carbon signal at 8C 101.0 (C-1'-). The two double doublet signals at 8H 3. 93 and 3. 72 correlated with carbon signal at 8C 60. 74 (C-6'-). The presence of sugar moiety in position 7 was proved by NOE spectrum (Fig. 1), which showed correlation between doublet at 8H 7. 81 (H-4) and the two signals at 8H 7. 03 (H-5) and doublet at 8H 6. 27 (H-3), correlation between singlet at 8H 7. 20 (H-8) and the doublet at 8H 4. 96 (H-1'-). Therefore, compound I was identified as cichoriin .
Cichoriin I, colorless oil, HRCIMS [M+1]+, m/z (rel. int.) 341 (80), C15H16O9, 178 [M+H-Glu. ]+ (75). IR ymaxKBr cm-1- 3295. 9, 2934. 5, 1595. 9, 1452. 5, 1125.8.: H-NMR (500 MHz, CD3OD). 13C NMR (500 MHz, CD3OD) 8: 160. 57 (C-2), 112. 97 (C-3), 143. 59 (C-4), 144. 17 (C-4a), 112. 65 (C-5), 113. 46 (C-6), 148. 81 (C-7), 103. 37 (C-8),
147. 79 (C-8a), 101. 00 (C-1'-), 75. 86 (C-2'-), 72. 28 (C-3'-), 73. 17 (C-4'-), 69. 81 (C-5'-), 60. 74 (C-6'-).
Cichoriin acetate Ia, Compound I (5 gm) was refluxed in 1 ml. of AC2O-CsH5N (1: 1) for 2h. The mixture was cooled to room temperature and extracted with CH2Cl2 to give the acetate Ia (3.5 gm). Colorless oil, IR ymaxKBr cm-1 2960. 5,
1588. 6, 1445. 8- HRCIMS m/z (rel int.) 551 [M+H]+ (80), C25H26O14- '-H-NMR spectral data (500 MHz, CD3OD).
Table 1. H-NMR spectral data of I-IV (500 MHz, CD3OD, TMS as int., standard, 8-values)*
I Ia+ II III IV
8h 8h 8h 8h 8h
3 6,27 (d, 9,5) 6,37 6,15 6,25 6,30
4 7,81 (d, 9,5) 7,65 7,75 7,90 7,65
5 7,03 (s) 7,05 6,74 6,80 6,86
8 7,20 (s) 7,20 6,93 7,25 6,93
1'- 4,96 (d, 7,5) 5,15
2'- 3,54 (dd, 7,5, 8,5) 5,35
3'- 3,50 (dd, 8,5, 9,0) 5,37
4'- 3,41 (dd, 9,0, 9,0) 5,36
5'- 3,51 (ddd, 3,0, 5,0, 9,0) 3,95
6'- 3,93 (dd, 12,0, 3,0) 4,35
3,72 (dd, 12,0, 3,0) 4,22
OMe 3,85 (s) 4,00 (s)
*Homonuclear 1H-1H COSY spectra were also used for these assignments. + OAc, 2. 04, 2. 07, 2. 08, 2. 14 and 2. 29.
The structures of the four compounds were elucidated as follows:
Esculetin II, yellowish brown oil, HRCIMS [M+1]+, m/z (rel. int.) 179 (95), C9H6O4. IR ymaxKBr cm-1- 3286. 5,
2922. 5, 1590. 9, 1450.5. 1HNMR (500 MHz, CD3OD). Scopoletin III, grayish oil, HRCIMS [M+1]+, m/z (rel. int.) 193 (90), C10H8O4. IR ymaxKBr cm-1- 3230. 5, 2900. 6, 1700. 5, 1452.5. '-HNMR (500 MHz, CD3OD) see table 1. Isoscopoletin IV, grayish oil, HRCIMS [M+1]+, m/z (rel. int.) 193 (93), Ci0H8O4. IR ymaxKBr cm-1- 3235. 5, 2905. 6,
1700. 6, 1450.5. '-HNMR (500 MHz, CD3OD). These cmpounds are explained instrumentally in Table 1 and structures are illustrated in Fig 1 and 2.
Acetylation of a portion of compound I gives the acetylated derivative Ia. HRCIMS provides a molecular ion peak [M+1]+ at m/z 551 corresponding to C25H26Oi4. The '-H-NMR spectrum revealed the five acetyl signals at 8H 2. 04, 2. 07, 2. 08, 2. 14 and 2. 29. The protons of the sugar and coumarin moieties were determined by 1H-1H COSY and given in table 1.
HRCIMS of compound II showed the molecular ion peak [M+1]+ at m/z 179 in accord with the molecular formula C9H6O4. 1H-NMR spectrum of II showed presence of two singlet signals at 8H 6. 93 (H-8) and 6. 74 (H-5) and the two doublets at SH 7. 75 (H-4, J = 9.5 Hz) and 6. 15 (H-3, J = 9.5 Hz). Therefore compound II was identified as esculetin.
The IR spectrum of III displayed absorption bonds characteristic of carbonyl group (1700 cm-1, C=O). The HRCIMS showed the molecular ion peak [M+1]+ at m/z 193 in accord with the molecular formula C10H8O4. The 1H-NMR spectrum of compound III revealed the presence of two doublets at SH 7. 90 (H-4, J = 6.0 Hz) and 6. 25 (H-3, J = 6.0 Hz). The two singlet signals appeared at SH 6. 80 and 7. 25 were assigned for the two protons H-5 and H-8, respectively. The difference between compound II and III was the presence of singlet signal at SH 3. 85, which assigned for a methoxy group. Therefore, compound III was identified as scopoletin.
1H-NMR spectrum of compound IV was close to compound III. The difference in the chemical shifts of the signals suggested that compound IV was isomer of compound III (isoscopoletin), H-8 of compound III appeared as singlet at SH 7. 25, whereas, H-8 of compound IV appeared as singlet at SH 6. 93. Also, few differences in the chemical shifts for H-5, H-3 and H-4 were observed, table 1. The HRCIMS which revealed a molecular ion peak [M+1]+ at m/z 193 which identical with the molecular formula C10H8O4.
Fig.1. NOE correlations compound 1
R1 = H R2 = H
R1 = Me R2 = H
R1 = H R2 = Me
Fig. 2. The structure of the isolated products
Results of antibacterial screening
In vitro, screening experiments for antibacterial activities of compounds I-IV was subjected to biological testing. To substantiate the antibacterial results, we screened compounds against an assortment of two Gram-positive bacteria (Bacillus cereus, Staphylococcus aureus) and Gram-negative bacteria (Serratia Sp., Pseudomonos Sp., Escherichia
coli) using ampicillin and amoxillin as a reference standard. The minimum inhibitory concentrations (MICs, ^g/ml) were determined using standard agar dilution method . The MIC value is summarized in Table 2.
From the obtained data, it is clear that compounds I-IV posses higher activity against Gram-positive strain, par-ticulerly Bacillus cereus. On the contrary, Gram-negative strains not affected at tested concentrations as shown in Table 2. Our results are in agreement with those reported earliear by Joklik et al. , they reported that some antibiotics such as ampicillin and amoxillin have been developed as inhibitors of cell wall synthesis of bacterial cell. So, compounds I-IV has the common structural feature of penicillins exhibit antibacterial activities.
Table 2. Antimicrobial activities of montanone (Dry DMSO as solvent):
Test organism F IF IIF IV Ampicillind Amoxillind
Gram- Positive Strain
Bacillus cereus 10a 10a 10a 10a 10a Na
18b 18b 18b 18b
Staphylococcus aureus Na Na Na Na 8a Na
5b 5b 5b 5b
Serratia sp Na Na Na Na 11a 13a
Nb Nb Nb Nb
Pseudomonas sp. Na Na Na Na 11a 13a
Nb Nb Nb Nb
Escherichia coli Na Na Na Na 11a 13a
Nb Nb Nb Nb
a Values show the zone of inhibition in mm.- conc. of the samples was 200 |ig/ml b Values show the zone of inhibition in mm.- conc. of the samples was 400|ig/ml c Data are the mean of five measurements with neglected standard errors. d Refference antibiotics were carried out at 200 |ig/ml only.
N = No effect
The authers are grateful to Industrial Education College, Beni Suif, for all facilities present to finish this work. Also grateful to Dr. Al-Domany R. A. Microbiology Department, Faculty of Pharmacy, El-Minia University, El-Minia, Egypt for his help for investigating the antibacterial activity of the isolated products.
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