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Germacranolides from Anvillea radiata
Fitoterapia 75 (2004) 573 – 576 www.elsevier.com/locate/fitote
Germacranolides from Anvillea radiata B. El Hassanya, F. El Hanbalia, M. Akssiraa,*, F. Melloukia, A. Haidourb, A.F. Barrerob a
Laboratoire de Chimie Bioorganique et Analytique, UFR C35/97 F.S.T Universite´ Hassan II-Mohammedia, BP 146, 20800 Mohammedia, Morocco b Instituto de Biotecnologia, Departamento de Quimica Organica, Facultad de Ciencias, Campus Fuentenueva s/n, 18071 Granada, Spain Received 10 March 2004; accepted 4 June 2004 Available online 6 August 2004
Abstract The aerial parts of Anvillea radiata yielded a new germacranolide, 8a,9a-epoxyparthenolide (3), together with two known compounds, 9a-hydroxyparthenolide (1) and parthenolid-9-one (2). The structures of the compounds were elucidated from IR, HRMS, 1H and 13C-NMR, COSY, HETCOR, HMBC and HOHAHA spectral data. The major component 1 was tested for its cytotoxicity and antibacterial activity. D 2004 Published by Elsevier B.V. Keywords: Anvillea radiata; 8a,9a-Epoxyparthenolide; Cytotoxicity; Antibacterial activity
1. Introduction In our continuing research on the study of different Moroccan endemic species, we have investigated the aerial parts of Anvillea radiata Coss. and Dur. and its biological activity. A. radiata is a wild plant predominantly distributed in steppes of north Africa (Morocco and Algeria). The plant is used in the folk medicine as excellent heating, for the treatment of dysentery, gastric–intestinal disorders [1] and has been reported to have hypoglycemic activity [2]. * Corresponding author. Tel.: +212 23 31 53 52; fax: +212 23 31 53 53. E-mail address: [email protected] (M. Akssira). 0367-326X/$ - see front matter D 2004 Published by Elsevier B.V. doi:10.1016/j.fitote.2004.06.003
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B. El Hassany et al. / Fitoterapia 75 (2004) 573–576
The phytochemistry of the genus has received less attention. From A. garcini, flavones [3] and parthenolide derivatives [4–6] were reported. These sesquiterpenes lactones have also been isolated previously from Matricaria suffructicasa var. Leptoloba [7] Anthemis cretica [8] and A. cupaniana [9]. An investigation of the A. radiata gave a new germacranolide 3 in addition to the known 9a-hydroxyparthenolide (1) and parthenolid-9-one (2) [4]. The structures were elucidated by combined 2D-NMR experiences.
2. Experimental 2.1. Plant material A. radiata (Asteraceae) was collected near Ouarzazate, South Morocco, in April 1999 and authenticated by Dr. A. Ouyahyia, Scientific Institute, Rabat, Morocco where a voucher specimen was deposited in the Herbarium. 2.2. Extraction and isolation The air-dried plant material (300 g) was successively extracted with hexane, chloroform and MeOH. The CHCl3 extract (30 g) was purified by treatment with MeOH/H2O at 4 8C. A 20 g of purified CHCl3 extract was Si–gel CC eluting with CH2Cl2 and CH2Cl2–EtOAc. Compound 1 (2.15 g) was obtained by recrystallization from the less polar fractions eluted with CH2Cl2–EtOAc (7:3). Si–gel CC of the same fraction eluted with hexane–CH2Cl2– EtOAc (3:5:2) gave 3 (27 mg). From the medium polar fraction 2 (56 mg) was isolated by recrystallization with ether. 8a,9a-Epoxyparthenolide (3), white powder; mp 227 8C (ether); [a]D25+55.58 (c 0.05, CHCl3); UVmax (CHCl3): 242 nm; IR bands (KBr): 1770(CMO) cm 1; HR-FABMS [M+Na]+, m/z 285.1102; calc. 285.1102. 1 H- and 13C-NMR data reported in Table 1 compared with 1H- and 13C-NMR data of the compounds 1 and 2. 2.3. Cytotoxic and antibacterial activities of 1 The cytotoxic activity of 1 was tested against five human cancer cell lines A 549, H 116, PSN 1, T98G, SKBR3 in accordance with the standard procedures of NCI [10].
B. El Hassany et al. / Fitoterapia 75 (2004) 573–576
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Table 1 NMR spectral data of compounds 1, 3 (CDCl3) and 2 (acetone-d6)a Position
1
2
dH 1 2a 2h 3a 3h 4 5 6 7 8a 8h 9h 10 11 12 13 13V 14 15 OH
5.55 2.18 2.42 1.21 2.09
ddd brd ddd ddd ddd
2.70 3.80 3.35 1.87 2.30 4.27
d t dddd dd ddd
5.61 6.21 1.63 1.23 2.65
d d s s br
3
dC
dH
121.61 d
6.44 2.48 2.65 1.32 2.29
t m m dt ddd
2.82 4.82 2.99 3.50 2.67
d t dddd dd dd
23.45 t 36.18 61.55 66.49 82.44 37.55
t s d d d
37.52 t 71.11 d 137.41 s 139.69 s 169.73 s 121.28 t 16.37 q 17.22 q
6.19 5.77 1.89 1.53
d d s s
dC
dH
140.15 d
5.84 2.42 2.64 1.58 2.32
brt brdd brddd ddd dddd
2.75 4.20 2.88 3.22
d t brs dd
23.94 t 35.99 63.38 65.97 81.83 44.59
t s d d d
40.36 203.43 138.78 139.63 168.94
t s s s s
119.98 t 12.57 q 17.18 q
dC
3.84 brd
6.09 6.52 1.84 1.52
d d s s
127.18 d 21.02 t 34.48 61.33 65.66 75.33 47.31
t s d d d
57.44 57.03 131.62 137.33 168.47
d d s s s
123.38 t 14.45 q 19.38 q
Complete assignments of the proton signals were determined from HMQC, HMBC and NOESY correlations. Multiplicities of the carbon signals were determined by DEPT pulse sequence. a 400 MHz for 1H and 100.577 MHz for 13C.
Antimicrobial activity of 1 was carried out by the disc diffusion method [11]. Microorganisms used: Bacillus cereus (IPL 58605), Streptococcus C (IPT 2-035), Proteus vulgaris (CIP 58605); Enterococcus faecalis (CIP 103214), Escherichia coli (CIP 54127) and Pseudomonas aeroginosa (CIP A 22) were supplied by the Pasteur Institute (Casablanca).
3. Results and discussion The CHCl3 extract of the aerial part of A. radiata afforded in addition to the two known germacranolides 1 and 2, a new partenolide 3. According to the spectral data, all of these compounds possessed the same 4a,5h-epoxygermacrene-6a,7h-olide skeleton. Compound 3 presented the molecular formula C15H18O4 as deduced from its HRFABMS ([M+Na]+, m/z 285.1103). The significant band in the IR spectrum at 1770 cm 1 is tipical for an a,h unsaturated g-lactones. The 1H and 13C-NMR spectral data of 3 (Table 1) showed general features similar to those of 1. The major difference was at C-8 and C-9, the 1H-NMR spectrum of 3 showing the presence of signals at d 3.22 (dd, J 4, 7.3 Hz) and d 3.84 (br d, J 4 Hz) corresponding to H-8h and H-9h while in 1 the methylene group at C-8 showed signals at d 1.87 (dd, J 8, 14.5 Hz) and d 2.3 (ddd, J 1, 5.6, 14.5 Hz) for H-8a and H-8h, respectively. The COSYDQF showed also that H-8
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B. El Hassany et al. / Fitoterapia 75 (2004) 573–576
Table 2 Antibacterial activity of 9a-hydroxyparthenolide (1)a Microorganisms Bacillus cereus Streptococcus C Enterococcus faecalis Escherichia coli Proteus vulgaris Pseudomonas aeroginosa
P
1 25 Ag/disc
50 Ag/disc
100 Ag/disc
8 6.5 6.5 – – –
10 7 7 9 8.5 –
14 8 7 9 9 –
13 19 13 16 9 9
–: No inhibition; P: penicillin G 10 UI. a Inhibition zone diameter measured in mm, the well diameter of 6 mm being included.
and H-9 are vicinal. The MS data, the absence of the hydroxyl absorption in IR and the chemical shifts in 13C- NMR of C-8 and C-9 stated the structure of compound 3 as 8a,9a-epoxyparthenolide. The relative configuration of carbons 8 and 9 were determined by NOE-difference experiments. Compound 1 demonstrated significant cytotoxic activity against five human cancer lines (IC50=2 Ag/ml against A 549, H 116, PSN 1 and SKBR 3 and IC50N5 Ag/ml against T98G). Table 2 shows that 9a-hydroxyparthenolide, at concentrations of 50 and 100 Ag/ disc, inhibited the growth of most of the tested microorganisms. However, the activity shown against E. coli is the most relevant when correlated with traditional use of the plant for the treatment of dysentery.
Acknowledgements This work was supported by grant from the ministry ESRSFC of the Moroccan Government (PROTARS P2T2/07) and from the Junta de Andalucı´a (Consejerı´a de Presidencia, Programa de Cooperacio´n Andalucı´a-Marruecos). We warmly thank Dr. A. Ouyahya for the identification of plant material.
References [1] Bellakhdar J. La pharmacope´e Marocaine traditionnelle. Ed Ibis press; 1997. [2] Al-yahya MA, Al-Meshal IA, Mossa JS, Al-Badr AA, Tariq M. Saudi Plants: A Phytochemical and Biological Approach. Riadh7 King Abdul Aziz City for Science and Technology (KACST); 1990. [3] Ulubelen A, Mabry TJ, Aynekdin Y. J Nat Prod 1979;42:624. [4] Sattar EA, Galal AM, Mossa GS. J Nat Prod 1996;59:403. [5] Tyson RL, Chang CJ, Melaughin JL, Aynehchi Y, Cassady JM. Experientia 1981;37:441. [6] Rustaiyan A, Dabiri M, Jakupovic J. Phytochemistry 1986;25:1229. [7] Bohlmann F, Zdero C. Chem Ber 1975;108:437. [8] Bohlmann F, Zdero C. Chem Ber 1975;108:1902. [9] Bruno M, Diaz JG, Herz W. Phytochemistry 1991;30:3458. [10] Monks A, Scrudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, et al. J Natl Cancer Inst 1991;83:757. [11] Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolke RH. Manual of Clinical Microbiology. 6th ed. Washington, (DC)7 ASM; 1995.
Germacranolides from Anvillea radiata B. El Hassanya, F. El Hanbalia, M. Akssiraa,*, F. Melloukia, A. Haidourb, A.F. Barrerob a
Laboratoire de Chimie Bioorganique et Analytique, UFR C35/97 F.S.T Universite´ Hassan II-Mohammedia, BP 146, 20800 Mohammedia, Morocco b Instituto de Biotecnologia, Departamento de Quimica Organica, Facultad de Ciencias, Campus Fuentenueva s/n, 18071 Granada, Spain Received 10 March 2004; accepted 4 June 2004 Available online 6 August 2004
Abstract The aerial parts of Anvillea radiata yielded a new germacranolide, 8a,9a-epoxyparthenolide (3), together with two known compounds, 9a-hydroxyparthenolide (1) and parthenolid-9-one (2). The structures of the compounds were elucidated from IR, HRMS, 1H and 13C-NMR, COSY, HETCOR, HMBC and HOHAHA spectral data. The major component 1 was tested for its cytotoxicity and antibacterial activity. D 2004 Published by Elsevier B.V. Keywords: Anvillea radiata; 8a,9a-Epoxyparthenolide; Cytotoxicity; Antibacterial activity
1. Introduction In our continuing research on the study of different Moroccan endemic species, we have investigated the aerial parts of Anvillea radiata Coss. and Dur. and its biological activity. A. radiata is a wild plant predominantly distributed in steppes of north Africa (Morocco and Algeria). The plant is used in the folk medicine as excellent heating, for the treatment of dysentery, gastric–intestinal disorders [1] and has been reported to have hypoglycemic activity [2]. * Corresponding author. Tel.: +212 23 31 53 52; fax: +212 23 31 53 53. E-mail address: [email protected] (M. Akssira). 0367-326X/$ - see front matter D 2004 Published by Elsevier B.V. doi:10.1016/j.fitote.2004.06.003
574
B. El Hassany et al. / Fitoterapia 75 (2004) 573–576
The phytochemistry of the genus has received less attention. From A. garcini, flavones [3] and parthenolide derivatives [4–6] were reported. These sesquiterpenes lactones have also been isolated previously from Matricaria suffructicasa var. Leptoloba [7] Anthemis cretica [8] and A. cupaniana [9]. An investigation of the A. radiata gave a new germacranolide 3 in addition to the known 9a-hydroxyparthenolide (1) and parthenolid-9-one (2) [4]. The structures were elucidated by combined 2D-NMR experiences.
2. Experimental 2.1. Plant material A. radiata (Asteraceae) was collected near Ouarzazate, South Morocco, in April 1999 and authenticated by Dr. A. Ouyahyia, Scientific Institute, Rabat, Morocco where a voucher specimen was deposited in the Herbarium. 2.2. Extraction and isolation The air-dried plant material (300 g) was successively extracted with hexane, chloroform and MeOH. The CHCl3 extract (30 g) was purified by treatment with MeOH/H2O at 4 8C. A 20 g of purified CHCl3 extract was Si–gel CC eluting with CH2Cl2 and CH2Cl2–EtOAc. Compound 1 (2.15 g) was obtained by recrystallization from the less polar fractions eluted with CH2Cl2–EtOAc (7:3). Si–gel CC of the same fraction eluted with hexane–CH2Cl2– EtOAc (3:5:2) gave 3 (27 mg). From the medium polar fraction 2 (56 mg) was isolated by recrystallization with ether. 8a,9a-Epoxyparthenolide (3), white powder; mp 227 8C (ether); [a]D25+55.58 (c 0.05, CHCl3); UVmax (CHCl3): 242 nm; IR bands (KBr): 1770(CMO) cm 1; HR-FABMS [M+Na]+, m/z 285.1102; calc. 285.1102. 1 H- and 13C-NMR data reported in Table 1 compared with 1H- and 13C-NMR data of the compounds 1 and 2. 2.3. Cytotoxic and antibacterial activities of 1 The cytotoxic activity of 1 was tested against five human cancer cell lines A 549, H 116, PSN 1, T98G, SKBR3 in accordance with the standard procedures of NCI [10].
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Table 1 NMR spectral data of compounds 1, 3 (CDCl3) and 2 (acetone-d6)a Position
1
2
dH 1 2a 2h 3a 3h 4 5 6 7 8a 8h 9h 10 11 12 13 13V 14 15 OH
5.55 2.18 2.42 1.21 2.09
ddd brd ddd ddd ddd
2.70 3.80 3.35 1.87 2.30 4.27
d t dddd dd ddd
5.61 6.21 1.63 1.23 2.65
d d s s br
3
dC
dH
121.61 d
6.44 2.48 2.65 1.32 2.29
t m m dt ddd
2.82 4.82 2.99 3.50 2.67
d t dddd dd dd
23.45 t 36.18 61.55 66.49 82.44 37.55
t s d d d
37.52 t 71.11 d 137.41 s 139.69 s 169.73 s 121.28 t 16.37 q 17.22 q
6.19 5.77 1.89 1.53
d d s s
dC
dH
140.15 d
5.84 2.42 2.64 1.58 2.32
brt brdd brddd ddd dddd
2.75 4.20 2.88 3.22
d t brs dd
23.94 t 35.99 63.38 65.97 81.83 44.59
t s d d d
40.36 203.43 138.78 139.63 168.94
t s s s s
119.98 t 12.57 q 17.18 q
dC
3.84 brd
6.09 6.52 1.84 1.52
d d s s
127.18 d 21.02 t 34.48 61.33 65.66 75.33 47.31
t s d d d
57.44 57.03 131.62 137.33 168.47
d d s s s
123.38 t 14.45 q 19.38 q
Complete assignments of the proton signals were determined from HMQC, HMBC and NOESY correlations. Multiplicities of the carbon signals were determined by DEPT pulse sequence. a 400 MHz for 1H and 100.577 MHz for 13C.
Antimicrobial activity of 1 was carried out by the disc diffusion method [11]. Microorganisms used: Bacillus cereus (IPL 58605), Streptococcus C (IPT 2-035), Proteus vulgaris (CIP 58605); Enterococcus faecalis (CIP 103214), Escherichia coli (CIP 54127) and Pseudomonas aeroginosa (CIP A 22) were supplied by the Pasteur Institute (Casablanca).
3. Results and discussion The CHCl3 extract of the aerial part of A. radiata afforded in addition to the two known germacranolides 1 and 2, a new partenolide 3. According to the spectral data, all of these compounds possessed the same 4a,5h-epoxygermacrene-6a,7h-olide skeleton. Compound 3 presented the molecular formula C15H18O4 as deduced from its HRFABMS ([M+Na]+, m/z 285.1103). The significant band in the IR spectrum at 1770 cm 1 is tipical for an a,h unsaturated g-lactones. The 1H and 13C-NMR spectral data of 3 (Table 1) showed general features similar to those of 1. The major difference was at C-8 and C-9, the 1H-NMR spectrum of 3 showing the presence of signals at d 3.22 (dd, J 4, 7.3 Hz) and d 3.84 (br d, J 4 Hz) corresponding to H-8h and H-9h while in 1 the methylene group at C-8 showed signals at d 1.87 (dd, J 8, 14.5 Hz) and d 2.3 (ddd, J 1, 5.6, 14.5 Hz) for H-8a and H-8h, respectively. The COSYDQF showed also that H-8
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B. El Hassany et al. / Fitoterapia 75 (2004) 573–576
Table 2 Antibacterial activity of 9a-hydroxyparthenolide (1)a Microorganisms Bacillus cereus Streptococcus C Enterococcus faecalis Escherichia coli Proteus vulgaris Pseudomonas aeroginosa
P
1 25 Ag/disc
50 Ag/disc
100 Ag/disc
8 6.5 6.5 – – –
10 7 7 9 8.5 –
14 8 7 9 9 –
13 19 13 16 9 9
–: No inhibition; P: penicillin G 10 UI. a Inhibition zone diameter measured in mm, the well diameter of 6 mm being included.
and H-9 are vicinal. The MS data, the absence of the hydroxyl absorption in IR and the chemical shifts in 13C- NMR of C-8 and C-9 stated the structure of compound 3 as 8a,9a-epoxyparthenolide. The relative configuration of carbons 8 and 9 were determined by NOE-difference experiments. Compound 1 demonstrated significant cytotoxic activity against five human cancer lines (IC50=2 Ag/ml against A 549, H 116, PSN 1 and SKBR 3 and IC50N5 Ag/ml against T98G). Table 2 shows that 9a-hydroxyparthenolide, at concentrations of 50 and 100 Ag/ disc, inhibited the growth of most of the tested microorganisms. However, the activity shown against E. coli is the most relevant when correlated with traditional use of the plant for the treatment of dysentery.
Acknowledgements This work was supported by grant from the ministry ESRSFC of the Moroccan Government (PROTARS P2T2/07) and from the Junta de Andalucı´a (Consejerı´a de Presidencia, Programa de Cooperacio´n Andalucı´a-Marruecos). We warmly thank Dr. A. Ouyahya for the identification of plant material.
References [1] Bellakhdar J. La pharmacope´e Marocaine traditionnelle. Ed Ibis press; 1997. [2] Al-yahya MA, Al-Meshal IA, Mossa JS, Al-Badr AA, Tariq M. Saudi Plants: A Phytochemical and Biological Approach. Riadh7 King Abdul Aziz City for Science and Technology (KACST); 1990. [3] Ulubelen A, Mabry TJ, Aynekdin Y. J Nat Prod 1979;42:624. [4] Sattar EA, Galal AM, Mossa GS. J Nat Prod 1996;59:403. [5] Tyson RL, Chang CJ, Melaughin JL, Aynehchi Y, Cassady JM. Experientia 1981;37:441. [6] Rustaiyan A, Dabiri M, Jakupovic J. Phytochemistry 1986;25:1229. [7] Bohlmann F, Zdero C. Chem Ber 1975;108:437. [8] Bohlmann F, Zdero C. Chem Ber 1975;108:1902. [9] Bruno M, Diaz JG, Herz W. Phytochemistry 1991;30:3458. [10] Monks A, Scrudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, et al. J Natl Cancer Inst 1991;83:757. [11] Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolke RH. Manual of Clinical Microbiology. 6th ed. Washington, (DC)7 ASM; 1995.