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Ovalifolone A and B: New friedelane derivatives from Garcinia ovalifolia
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PHYTOL 484 1–5 Phytochemistry Letters xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol 1 2 3 4 5 6
Ovalifolone A and B: New friedelane derivatives from Garcinia ovalifolia Q1 Alain
Meli Lannang a,b,*, Bodrix Solitaire Noudou c, Norbert Sewald b
a
Department of Chemistry, Higher Teachers’ Training College, University of Maroua, P.O. Box 55, Maroua, Cameroon Department of Chemistry, Organic and Bioorganic Chemistry, Bielefeld University, P.O. Box 100131, 33501 Bielefeld, Germany c Department of Organic Chemistry, Faculty of Science, University of Yaounde´ I, P.O. Box 812 Yaounde´, Cameroon b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 November 2012 Received in revised form 20 December 2012 Accepted 21 December 2012 Available online xxx
Two new friedelane type triterpenoids, ovalifolone A (1) and ovalifolone B (2), together with eight known compounds were isolated from the stem bark of Garcinia ovalifolia. Their structures were established on the basis of mass spectrometric, NMR data and by the comparison with literature data. Some of the isolated compounds were evaluated for their cytotoxicity and antibacterial activities and garcinane showed interesting activity against Artemia salina with LD50 value of 2.69 mg/mL. ß 2013 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Keywords: Garcina ovalifolia Ovalifolone Cytotoxicity Antibacterial activity
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Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . Experimental . . . . . . . . . . . . . . . . . . . . General . . . . . . . . . . . . . . . . . . . 3.1. Plant material . . . . . . . . . . . . . . 3.2. Extraction and isolation . . . . . . 3.3. Paper disk agar diffusion assay 3.4. Cytotoxicity assay . . . . . . . . . . . 3.5. 3.6. Determination of LD50 values. . Ovalifolone A (1) . . . . . . . . . . . . 3.7. Ovalifolone B (2) . . . . . . . . . . . . 3.8. Acknowledgements . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .
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000 000 000 000 000 000 000 000 000 000 000 000 000
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1. Introduction
11 12 13 14
The genus Garcinia (Guttiferae) is known to produce a variety of biologically active metabolites such as polyisoprenylated benzophenones (Meli Lannang et al., 2010; Gustafson et al., 1992), xanthones (Louh et al., 2008) as well as triterpenoids (Nguyen
* Corresponding author at: Higher Teachers’ Training College, Department of Chemistry, University of Maroua, P.O. Box 55, Maroua, Cameroon. Tel.: +237 77 53 48 30; fax: +237 22 22 91 16. E-mail addresses: [email protected], [email protected] (A.M. Lannang).
et al., 2011; Meli Lannang et al., 2008). In the continuation of our search for bioactive substances from Garcinia species, we have investigated the mixture of hexane and dichloromethane extract of the stem bark of Garcinia ovalifolia. G. ovalifolia is a tree up to 10– 15 m on high, with yellow sticky latex, generally distributed in fringing forests and riverbanks in West and central Africa (Gustafson et al., 1992). We describe herein the isolation and structure elucidation of minor constituents from G. ovalifolia, which included two new friedelane triterpene derivatives, ovalifolone A (1), and B (2) (Fig. 1) together with eight known compounds 3–10. Some of these isolated compounds were tested for cytotoxicity and antibacterial activities.
1874-3900/$ – see front matter ß 2013 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe. http://dx.doi.org/10.1016/j.phytol.2012.12.010
Please cite this article in press as: Lannang, A.M., et al., Ovalifolone A and B: New friedelane derivatives from Garcinia ovalifolia. Phytochem. Lett. (2013), http://dx.doi.org/10.1016/j.phytol.2012.12.010
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PHYTOL 484 1–5 A.M. Lannang et al. / Phytochemistry Letters xxx (2013) xxx–xxx
2
Fig. 2. Selected HMBC correlation of compounds 1 and 2.
Fig. 1. Structures of compounds 1 and 2.
27
2. Results and discussion
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The mixture of hexane and dichloromethane extract from the stem bark of G. ovalifolia was subjected to repeated column chromatography and 10 compounds were isolated: two new friedelane derivatives, ovalifolone A (1) and ovalifolone B (2); and eight known secondary metabolites, namely friedelin (3) (Ngouamegne et al., 2008), endodesmiadiol (4) (Ngouamegne et al., 2008), canophyllol (5) (Ngouamegne et al., 2008), canophyllal (6) (Ngouamegne et al., 2008), garcinane (7) (Meli Lannang et al., 2008), gallic acid (8) (Sudjaroen et al., 2012), isoxanthochimol (9) (Louh et al., 2008) and 3-methoxycheffouxanthone (10) (Meli Lannang et al., 2010). Compound 1 was obtained as a white amorphous solid. Its molecular formula was established as C30H48O3 from HREIMS data (m/z 456.35980 for [M]+), indicating seven degrees of unsaturation. The positive response to the Liebermann-Burchard test suggested the triterpenic nature of 1. The IR spectrum showed absorption bands characteristic of OH (3420 cm1) and carbonyl (1699 cm1) groups. Analysis of the 13C NMR (Table 1) in combination with
Table 1 NMR (1H: 500 MHz, Position
13
C: 125 MHz) data of compounds 1–2. Compound 1, CDCl3 1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 3-OH
DEPT and HMQC experiments revealed the presence of 30 signals including seven methyls (dc 32.8, 34.2, 19.0, 18.8, 18.8, 17.8 and 17.8), eleven sp3 methylenes (dc 38.3, 34.5, 34.4, 33.3, 32.3, 31.2, 31.1, 29.8, 29.0 and 17.9) among which one carbinol (dc 68.0), tree sp3 methines (dc 55.6, 52.0 and 39.4) and nine quaternary carbons (dc 142.5, 140.7, 39.5, 39.3, 36.7, 35.1 and 28.1) among which one carbonyl group (dc 194.9). The 1H NMR spectrum (Table 1) showed seven methyl singlets. These data were in agreement with the structure of the friedelane skeleton, ascribing the signal at dH 1.83 (s) to H-23 (Ngouamegne et al., 2008). This last signal showed a HMQC correlations with the shielded methyl carbon at dc 10.3 and HMBC correlation with the quaternary carbons at dc 140.7 (C-4), 142.5 (C-3) and the methyl carbon at dc 17.8 (C-24), establishing the ketone group in ring A at C-2 and the double bond between C-3 and C-4. This was supported by the chemical shifts at dH 2.53 (Ha1) and 2.42 (Hb-1) showing HMQC correlations with the methylene carbon at dc 32.3 and HMBC correlations (Fig. 2) with quaternary carbons at dc 194.9 (C-2), 142.5 (C-3), 39.5 (C-5) and the methine carbon at dc 55.6 (C-10). A primary OH group was evident in the 1H NMR spectrum by two doublets located at dH 3.64 d (J = 15.5 Hz)
2.53 2.42 – – – – 1.94 1.43 1.43 – 1.83 1.46 1.87 – – 1.41 1.31 – 1.29 1.40 – 1.33 1.87 1.83 1.11 1.08 0.95 1.10 3.64 3.65 0.95 0.99 –
Compound 2, CDCl3 13
H (1H, dd, J = 17.6, 3.9 Hz) (1H, dd, J = 17.6, 13.9 Hz)
(1H, m); 1.46 (1H, m) (1H, m), 1.56 (1H, m) (1H, m) (1H, m) (2H, m) (2H, m)
(2H, m) (2H, m) (1H, m) (2H, m) (2H, (2H, (3H, (3H, (3H, (3H, (3H, (1H, (1H, (3H, (3H,
m) m) s) s) s) s) s) d, J = 15.5 Hz) d, J = 15.5 Hz) s) s)
C
32.3 194.9 142.5 140.7 39.5 38.3 17.9 52.0 36.7 55.6 34.5 29.0 39.3 39.4 31.1 31.2 35.1 39.4 34.4 28.1 33.3 29.8 10.3 17.8 18.8 19.0 18.8 68.0 34.2 32.8 –
1
13
H
2.55 2.42 – 3.80 1.32 – 1.09 1.41 1.33 – 1.34 1.30 1.31 – – 1.41 1.86 – 1.29 1.28 – 1.22 1.02 1.05 1.07 0.91 0.93 1.08 3.68
(1H, dd, J = 3.5; 13.8 Hz) (1H, t, J = 13.8 Hz) (1H, dd, J = 3.8; 11.6 Hz) (1H, m) (1H, m); 1.86 (1H, m) (1H, m); 1.51 (1H, m) (1H, m) (1H, m) (2H, m) (2H, m)
(1H, m); 1.48 (1H, m) (2H, m) (1H, m) (2H, m) (2H, (1H, (3H, (3H, (3H, (3H, (3H, (2H,
m) m); 1.32 (1H, m) d, J = 6.0 Hz) s) s) s) s) br s)
0.99 (3H, s) 1.00 (3H, s) 3.56 (OH, d, J = 3.8 Hz)
C
36.1 211.8 76.8 54.5 38.0 40.5 17.5 52.5 37.6 60.3 34.5 31.2 38.1 38.0 31.3 29.9 35.1 39.4 34.4 28.1 31.2 33.3 10.8 14.2 17.6 19.0 19.1 68.0 34.3 31.2 –
Please cite this article in press as: Lannang, A.M., et al., Ovalifolone A and B: New friedelane derivatives from Garcinia ovalifolia. Phytochem. Lett. (2013), http://dx.doi.org/10.1016/j.phytol.2012.12.010
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PHYTOL 484 1–5 A.M. Lannang et al. / Phytochemistry Letters xxx (2013) xxx–xxx
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Table 2 Determination of lethal dose LD50. Sample
10 mg/mL
5 mg/mL
2.5 mg/mL
1.25 mg/mL
0.02 mg/mL
LD50
7 Actinomycine D Berberine hydrochloride
100 100 n.m.
91 100 n.m.
45 100 n.m.
0 100 n.m.
n.m. 50 n.m.
2.69 mg/mL 0.02 mg/mL 26 mg/mL
Notes. Tests were carried out in duplicate. The mean percentage mortality was calculated for each concentration after the formula (A-N-B)Z1100, with A = number of dead larvas after 24 h; N = number of dead larvas before the sample was added B = number of dead larvas in the negative control after 24 h Z = total number of larvas. Mortality percentages were plotted against the logarithm of concentrations and LD50, at which 50% of the Artemia salina died, was determined from the graph. For Actinomycine D, LD50 Q3 directly resulted from the assay. The LD50 value of Berberine hydrochloride was taken from the literature (Saidnia et al., 2009). n.m. = not measured.
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98
and 3.65 (J = 15.5 Hz), which correspond to the carbinol methylene protons H-28. The presence of this group was supported by the signal of an oxygenated methylene carbon at dc 68.0 in the 13C NMR carbon spectrum. These carbinol protons showed long range correlations with the methine group at dc 39.4 (C-18), two methylene groups at dc 29.8 (C-22), 31.2 (C-16) and a quaternary carbon at dc 35.1 (C-17). The additional 1H and 13C NMR signals were similar to those reported for friedelane triterpene (Ngouamegne et al., 2008). Thus, compound 1 was established as 3, 28 dihydroxyfriedel-3-en-2-one (Fig. 1) and was trivially named ovalifolone A (Table 2). Compound 2 was isolated as colorless needles. Its IR spectrum exhibited bands of OH (3370 cm1) and carbonyl (1669 cm1) groups. The molecular formula was established as C30H50O3 from HREIMS data at (m/z 458.37520 for [M]+), indicating six degrees of insaturation. The positive response to the Liebermann-Burchard test suggested the triterpenic nature of compound 2. The 13C NMR spectrum (Table 1) showed 30 carbon signals, and the DEPT spectra indicated the presence of seven methyls, eleven methylenes including one carbinol (dc 68.0), five methines including one carbinol (dc 76.8), and seven quaternary carbons with one carbonyl at dc 211.8 ppm. The 1H NMR spectrum (Table 1) showed seven methyls signals, including six singlets (dH 1.08, 1.07, 1.00, 0.99, 0.93 and 0.91), and a doublet at dH 1.05 (J = 6.0 Hz), similar to those of endodesmiadol (Ngouamegne et al., 2008) The last signal was ascribed to H-23 since it showed a HMQC correlation with the shielded methyl carbon at dc 10.8 and crosspeaks HMBC with two methine carbon at dc 76.8 (C-3) and 54.5 (C-4), and with a quaternary carbon at dc 38.0 (C-5) establishing the hydroxyl group at C-3. The exchangeable proton (OH-3) resonated at dH 3.56 as a doublet (J = 3.8 Hz), showed long range correlations with the carbonyl group at dc 211.8 (C-2) and two methines at dc 76.8 (C-3) and (dc 54.5 (C-4) establishing a ketone group at C-2. The doublet of
Fig. 3. Selected NOESY and COSY of compound 2.
doublets at dH 3.85 (J = 3.8 and 11.6 Hz) corresponds to the proton 99 H-3. This proton was assigned to have b-orientation from the J 100 coupling constant and from NOESY experiments (Fig. 3), which 101 exhibited proximity to methyl C-23, methyl C-24 and H-1b. The 102 OH in position 3 was assigned unambiguously to have a- 103 orientation. The doublet of doublet at dH 2.55 (J = 13.8 and 104 3.2 Hz) and the broad triplet at dH 2.42 (J = 13.8 Hz) were assigned 105 to the methylene protons H-1a and H-1b, respectively. The 106 additional 1H and 13C NMR signals were similar to those of 107 friedelane (Ngouamegne et al., 2008). Thus, compound 2 was 108 established as 3a, 28 dihydroxyfriedelan-2-one (Fig. 1) and was 109 trivially named ovalifolone B. 110 Compounds 1, 2, 4, 5, 7 and 8 were tested for their biological 111 activity against various microbes by paper disk diffusion assay at 112 40 mg/paper disk, and against Artemia salina for cytotoxicity 113 assay at 10 mg/mL. Compound 7 responded positively in the 114 cytotoxicity assay and, the lethal dose LD50, which corresponds 115 to the concentration that killed 50% of the brine shrimps, was 116 determined to be 2.69 mg/mL. Other compounds did not show 117 LD50 values >10.0 mg/mL. Thus, compound 7 is more active than 118 to the well known anti cancer active alkaloid berberine 119 hydrochloride with LD50 = 26.0 mg/mL (Saidnia et al., 2012). Q2120 None of the tested samples showed activity against the bacteria 121 Staphylococcus aureus and Escherichia coli, the microfungi Candida 122 albicans and Mucor miehei and the plant pathogenic fungus 123 Rhizoctonia solani. Furthermore, compound 8 displayed moderate 124 activity against the plant pathogenic fungi Pythium ultimum 125 (14 mm). It should be noted that from a pharmacological point of 126 view, a good correlation between cytotoxic activities in the A. 127 salina test and antitumor, as well as antimalaria activities was 128 reported (Adoum, 2009). 129 3. Experimental
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3.1. General
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Optical rotations were measured in methanol solution on a JASCO digital polarimeter (model DIP-3600). The specific rotation is given in deg cm2 g1. IR spectra were recorded in CHCl3 on a JASCO A-302 IR spectrophotometer. The 1H, 13C, and 2D-NMR spectra were recorded on a Bruker AMX-500 spectrometer using CDCl3 as solvent. Homonuclear 1 H–1H connectivities were determined by using the COSY 458 experiment. One-bond 1 H–13C connectivities were determined by HMQC. Two- and three-bond 1 H–13C connectivities are determined by HMBC experiment. Proton chemical shifts are reported in d (ppm) with reference to the residual CDCl3 signal at d 7.26, and 13C NMR spectra are referenced to the central peak of CDCl3 at d 77.0. Coupling constants (J) were measured in Hz. The EIMS were recorded on a double-focusing mass spectrometer (Varian MAT 311A). HREIMS were recorded on a JEOL HX 110 mass spectrometer. Column chromatography was carried out with silica gel 60 (70–230 and 240–300 mesh sizes, E. Merck). Precoated silica gel TLC plates (E. Merck, F254) were used to
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Please cite this article in press as: Lannang, A.M., et al., Ovalifolone A and B: New friedelane derivatives from Garcinia ovalifolia. Phytochem. Lett. (2013), http://dx.doi.org/10.1016/j.phytol.2012.12.010
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check the purity of compounds, and ceric sulfate spray reagent was used for the visualization of compounds on TLC plates.
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3.2. Plant material
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The stem bark and leaves of Garcinia ovalifolia were collected at ‘‘Mont Kala’’ locality in Center Cameroon in April 2010 and identified by Mr. Nana Victor of the National Herbarium, Yaounde´, Cameroon, where a voucher specimen (ref. 55523/HNC) was deposited.
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3.3. Extraction and isolation
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The air-dried and powdered stem bark (4.5 kg) of Garcinia ovalifolia was separately extracted with MeOH at room temperature for 72 h. After evaporation under reduced pressure, 250.0 g of brown crude material were obtained. The crude extract (200 g) was suspended in aqueous MeOH (MeOH–H2O, 9:1, 2000 mL) and extracted with n-hexane (3 500 mL). The aqueous layer was then diluted with 60% MeOH and extracted with CH2Cl2 (3 500 mL), then with ethyl acetate. The extracts were concentrated under reduced pressure to afford 50.6, 60.8 and 25.0 g of n-hexane, dichloromethane and ethyl acetate extracts, respectively. The n-hexane and CH2Cl2 extracts were combined on the basis of TLC profile. This combined fraction was purified by column chromatography over silica gel 60 (230–400 mesh) and preparative TLC using a gradient system of n-hexane, CH2Cl2, ethyl acetate, and MeOH. All together, 210 subfractions (ca. 250 mL each) were collected and pooled on the basis of TLC analysis, leading to six main fractions (A–G). Fraction A (10.0 g) was the combination of subfractions 1–20, eluted with a mixture of n hexane–CH2Cl2 (8:2). Fraction B (10.0 g) was constituted of subfractions 21–49, eluted with a mixture of n -hexane–CH2Cl2 (6:4). Fraction C (10.0 g) was the combination of subfractions 50– 90, eluted with n -hexane–CH2Cl2 (1:1). Fraction D (10.0 g) was constituted of subfractions 91–122, eluted with pure CH2Cl2. Fraction E (10.0 g) was the combination of subfractions 123–152, eluted with CH2Cl 2–ethyl acetate (9:1). Fraction F (10.0 g) was constituted of subfractions 153–198, eluted with CH2Cl 2–ethyl acetate (8:2) and fraction G (9.0 g) constituted of subfraction 199–210, eluted with ethyl acetate–methanol (9:1). Fraction A was chromatographed over a silica gel 60C (20–40 mm) column with n -hexane–CH2Cl2 gradient. A total of 25 fractions of ca. 100 mL each were collected and combined on the basis of TLC profile. Subfractions 1–20 were further chromatographed on silica gel 60H (5–40 mm) with a mixture of n -hexane–CH2Cl2 (8:2) for elution to yield friedelan (3) (15.5 mg) and canophyllal (6) (8.5 mg). Fraction B was chromatographed over a silica gel 60C column with n -hexane–CH2Cl2 gradient. A total of 30 fractions of ca. 100 mL each were collected and combined on the basis of TLC. Subfractions 1–10 were further chromatographed over a silica gel 60H with a mixture of n -hexane–CH2Cl2 (4:1) to yield canophyllol (5) (10.0 mg), ovalifolone A (1) (12.0 mg), endodesmiadol (4) (7.5 mg), and gallic acid (8) (8.4 mg). Fraction C was chromatographed on a silica gel 60C column with n -hexane–CH2Cl2 gradient. All together, 35 fractions of ca. 100 mL each were collected and combined on the basis of TLC profile. Subfractions 21–35 were further chromatographed over a silica gel 60H with n hexane–CH2Cl2 (1:1) to yield ovalifolone B (2) (10.0 mg), garcinane (7) (6.0 mg), and 3-methylcheffouxanthone (10) (12.0 mg). Similarly, fraction G was chromatographed on a silica gel 60C column with n -hexane–CH2Cl2 (1:3) gradient. As a result, 20 fractions of ca. 100 mL each were collected and combined on the basis of TLC profile. Subfractions 8–15 were further chromatographed over silica gel 60H with n -hexane–CH2Cl2 (1:3) to yield isoxanthochimol (9) (40.0 mg).
3.4. Paper disk agar diffusion assay
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Agar test plates for bacteria and micro fungi are prepared as twolayer plates containing the microbial test strain in the uppermost layer (Maskey et al., 2002). Agar media are peptone agar for Bacillus subtilis and E. coli, Bacto nutrient agar for S. aureus, M2 agar for M. miehei and Sabouraud agar for C. albicans. Test plates for plant pathogen fungi R. solani, P. ultimum and Aphanomyces cochlioides are prepared as single layer plates of potato-dextrose agar (PDA). For bacteria and micro fungi, a few colonies of the test organism are picked with a wire loop from the original culture plate and introduced into a test tube containing 2 mL of fluid test medium. The tube is incubated for 12 h on a reciprocal shaker at 37 8C at 180 rpm. Petri dishes are filled with 25 mL of handwarm agar medium to give a depth of about 5 mm. The test strain suspension is 10-fold diluted with fluid test strain medium, mixed 1:4 with handwarm agar and 5 mL are poured on the first already stiff layer, to give a second 1 mm layer of so called ‘‘soft agar’’ due to less agar in this layer. For plant pathogen fungi, Petri dishes are filled with handwarm PDA agar to give a depth of about 5 mm and cooled down to become stiff. 1 cm 1 cm squares are cut from the growth front of well grown agar plates by a microbiologic hook and placed inverse onto the center of fresh plates. R. solani and P. ultimum plates are cultivated at 28 8C in the dark and A. cochlioides plates at room temperature (23 8C) in the dark until the diameter of the growing fungi measured around 6 cm. Above procedures are carried out under sterile conditions, i.e. media are autoclaved, agar plates and cultures are prepared in a clean bench, and wire loop and microbiologic hook are sterilized over the flame of a bunsen burner placed inside the clean bench. Six test samples and reference Cycloheximid are dissolved in DCM/MeOH 9:1, reference streptomycin sulfate in pure DMSO. 40 mL of a 1 mg/mL solution are applied on the disks. Solvents are evaporated for 1 h. Five paper disks impregnated with compound and one paper disk containing the corresponding reference are placed in a circle on the surface of each test agar plate with flamed forceps and gently pressed down to ensure contact. The plates containing bacteria and micro fungi are inverted and incubated for 12 h at 37 8C. Plates with plant pathogen fungi R. solani and P. ultimum are incubated for two to three days at 28 8C in the dark, and A. cochlioides at room temperature. The diameters of zones with complete inhibition of test strain growth are measured with a ruler and given in mm. The bacteria S. aureus, B. subtilis, E. coli and the microfungus C. albicans are Cameroonian clinical isolates; the microfungus M. miehei and the plant pathogen fungi originate from the Strain Collection Tu¨bingen of the Institute of Plant Pathology and Crop Protection, Go¨ttingen.
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3.5. Cytotoxicity assay
259
The A. salina toxicity assay is a simple, rapid and inexpensive bench top assay and considered as a useful tool for preliminary assessment of general toxicity (Meyer et al., 1982). A separation funnel is filled with 1 L of filtered artificial seawater and 200 mg of A. salina eggs and kept under permanent light and at constant temperature until the larva hatch. 990 mL of seawater containing 20 or more larvas are pipetted in each well of a 24-well microtiter plate. Dead larvae are recorded under a microscope. 10 mL of 1 mg/mL solution of each compounds in DMSO is added to make an end concentration of 10 mg/mL. The tests are carried out in duplicate. For negative control, 10 mL of DMSO and for positive control, 10 mL of a 1 mg/mL solution of actinomycin D in DMSO is used. After incubation for 24 h at permanent light and constant temperature, dead larvas are counted. The plates are kept in the refrigerator for 24 hours to
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immobilize the larvae. The total number of larvas is calculated for each well. The mortality in % is determined with the formula (A–N– B)Z1100, with A = number of dead larvas after 24 h; N = number of dead larvas before the addition of tested compound; B = number of dead larvas in the negative control; Z = total number of larvas. Anhydrous A. salina eggs were purchased in a German pet shop.
Acknowledgments
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A.M.L. would like to thank the Alexander von HumboldtStiftung for providing Georg Forster Fellowship for experienced researchers. This article is also dedicated to the memory of Professor David Lontsi who passed away on December 22, 2008.
308 309 310 311
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3.6. Determination of LD50 values
References
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282 283 284 285 286 287 288 289 290 291 292 293 294
Lethal dose LD50 of compound 7 active at 10 mg/mL in the cytotoxicity assay against A. salina was determined. The sample was dissolved in DMSO and serial diluted to give end concentrations of 10, 5, 2.5, 1.25, 0.625 mg/mL. Tests were carried out in duplicate. For the positive control Actinomycine D, the dilution line was extended to the end concentration of 0.006 mg/mL, since the compound is extremely high toxic and a low value was expected. The mean percentage mortality was calculated for each concentration after the formula (A–N–B)Z1100 (see above). Mortality percentages were plotted against the logarithm of concentrations and LD50, at which 50% of the A. salina died, was determined from the graph. For the positive control Actinomycine D, LD50 directly resulted from the calculation of mortalities.
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3.7. Ovalifolone A (1)
296 297 298 299 300
Amorphous powder, ½a25 D þ 1:8 ðc 0:03; acetoneÞ. IR (CHCl3): nmax 3450, 3100, 2943, 2360, 2009, 1733, 1699, 14921213, 1086 cm1. EI-MS: m/z 456 [M]+ (16), 425 (98), 426 (82), 289 (70), 137 (100). HREIMS: m/z 456.3598 (calc, 456.3603) for C30H48O3. 1H and 13C NMR data, see Table 1.
301
3.8. Ovalifolone B (2)
302 303 304 305 306
Colorless powder, mp: 236–237 8C. ½a25 D þ 6:6 ðc 0:05; acetoneÞ. IR (CHCl3): nmax 3648, 3350, 2940, 2363, 2057, 1792, 1704, 1473 cm1. EI-MS: m/z 458 [M]+ (10), 427 (80), 289 (49), 137 (100). HREIMS: m/z 458.3752 (calc, 458.3760) for C30H50O3. 1H and 13C NMR data, see Table 1.
Adoum, O.A., 2009. Determination of toxicity levels of some savannah plants using brine shrimp test (BST). Bayero J. Pure Appl. Sci. 2, 135–138. Gustafson, K.R., Blunt, J.W., Munro, M.H.G., Fuller, R.W., McKee, T.C., Cardellina II, J.H., McMahon, J.B., Cragg, G.M., Boyd, M.R., 1992. The guttiferones, HIV inhibitory agents from Symphonia globulifera, Garcinia livingstonei, Garcinia ovalifolia and Clusia rosea. Tetrahedron 48, 10093–10102. Louh, N.G., Meli Lannang, A., Mbazoa, D.C., Tangmouo, J.G., komguem, J., Castillo, P., Mofo, N.F., Naz, Q., Lontsi, D., Iqbal, M.C., Sondengam, B.L., 2008. Polyanxanthone A, B and C, three xanthones from the wood trunk of Garcinia polyantha Oliv. Phytochemistry 69, 1013–1017. Maskey, R.P., Ratnakar, N., Asolkar, E.K., Wagner-Do¨bler, I., Laatsch, H., 2002. Phytotoxic arylethylamides from limnic bacteria using a screening with microalgae. J. Antibiot. 55, 643–649. Meli Lannang, A., Louh, G.N., Biloa, B.M., Komguem, J., Mbazoa, C.D., Sondengam, B.L., Naesens, L., Pannecouque, C., Clercq, E.D., Ashry, E.S.H.E., 2010. Cytotoxicity of natural compounds isolated from the seed of Garcinia afzelii ENGL. (Guttiferae). Planta Med. 76, 708–712. Meli Lannang, A., Louh, N.G., Lontsi, D., Specht, S., Sarite, S.R., Flo¨rke, U., Hussain, H., Hoerauf, A., Krohn, K., 2008. Antimalarial compounds from the root bark of Garcinia polyantha Oliv. J. Antibiot. 61, 518–523. Meyer, B.N., Ferrigni, N.R., Putnam, J.E., Jacobsen, L.B., Nichols, D.E., McLaughlin, J.L., 1982. Brine shrimp: a convenient general bioassay for active plant constituents. Plant. Med. 45, 31–34. Ngouamegne, E.T., Fongang, R.S., Ngouela, S., Boyom, F.F., Rohmer, M., Tsamo, E., Gut, J., Rosenthal, P., 2008. Endodesmiadiol, a friedelane triterpenoid, and other antiplasmodial compounds from Endodesmia calophylloides. J. Chem. Pharm. Bull. 56, 374–377. Nguyen, H.D., Trinh, B.T., Tran, Q.N., Nguyen, H.D., Pham, H.D., Hansen, P.E., Duus, F., Connolly, J.D., Nguyen, L.H., 2011. Friedolanostane, friedocycloartane and benzophenone constituents of the bark and leaves of Garcinia benthami. Phytochemistry 72, 290–295. Saidnia, S., Gohari, A.R., Shahverdi, A.R., Permeh, P., Nasiri, M., Mollazadeh, K., Farahani, F., 2009. Biological activity of two red algae Gracilaria salicornia and Hypnea flagelliformis from Persian Gulf. Phcog. Res. 1, 428–430. Sudjaroen, Y., Hull, W.E., Erben, G., Wu¨rtele, G., Changbumrung, S., Ulrich, C.M., Owen, R.W., 2012. Isolation and characterization of ellagitannins as the major polyphenolic components of Longan (Dimocarpus longan Lour) seeds. Phytochemistry 77, 226–237.
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PHYTOL 484 1–5 Phytochemistry Letters xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol 1 2 3 4 5 6
Ovalifolone A and B: New friedelane derivatives from Garcinia ovalifolia Q1 Alain
Meli Lannang a,b,*, Bodrix Solitaire Noudou c, Norbert Sewald b
a
Department of Chemistry, Higher Teachers’ Training College, University of Maroua, P.O. Box 55, Maroua, Cameroon Department of Chemistry, Organic and Bioorganic Chemistry, Bielefeld University, P.O. Box 100131, 33501 Bielefeld, Germany c Department of Organic Chemistry, Faculty of Science, University of Yaounde´ I, P.O. Box 812 Yaounde´, Cameroon b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 November 2012 Received in revised form 20 December 2012 Accepted 21 December 2012 Available online xxx
Two new friedelane type triterpenoids, ovalifolone A (1) and ovalifolone B (2), together with eight known compounds were isolated from the stem bark of Garcinia ovalifolia. Their structures were established on the basis of mass spectrometric, NMR data and by the comparison with literature data. Some of the isolated compounds were evaluated for their cytotoxicity and antibacterial activities and garcinane showed interesting activity against Artemia salina with LD50 value of 2.69 mg/mL. ß 2013 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Keywords: Garcina ovalifolia Ovalifolone Cytotoxicity Antibacterial activity
7 8
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . Experimental . . . . . . . . . . . . . . . . . . . . General . . . . . . . . . . . . . . . . . . . 3.1. Plant material . . . . . . . . . . . . . . 3.2. Extraction and isolation . . . . . . 3.3. Paper disk agar diffusion assay 3.4. Cytotoxicity assay . . . . . . . . . . . 3.5. 3.6. Determination of LD50 values. . Ovalifolone A (1) . . . . . . . . . . . . 3.7. Ovalifolone B (2) . . . . . . . . . . . . 3.8. Acknowledgements . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .
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10
1. Introduction
11 12 13 14
The genus Garcinia (Guttiferae) is known to produce a variety of biologically active metabolites such as polyisoprenylated benzophenones (Meli Lannang et al., 2010; Gustafson et al., 1992), xanthones (Louh et al., 2008) as well as triterpenoids (Nguyen
* Corresponding author at: Higher Teachers’ Training College, Department of Chemistry, University of Maroua, P.O. Box 55, Maroua, Cameroon. Tel.: +237 77 53 48 30; fax: +237 22 22 91 16. E-mail addresses: [email protected], [email protected] (A.M. Lannang).
et al., 2011; Meli Lannang et al., 2008). In the continuation of our search for bioactive substances from Garcinia species, we have investigated the mixture of hexane and dichloromethane extract of the stem bark of Garcinia ovalifolia. G. ovalifolia is a tree up to 10– 15 m on high, with yellow sticky latex, generally distributed in fringing forests and riverbanks in West and central Africa (Gustafson et al., 1992). We describe herein the isolation and structure elucidation of minor constituents from G. ovalifolia, which included two new friedelane triterpene derivatives, ovalifolone A (1), and B (2) (Fig. 1) together with eight known compounds 3–10. Some of these isolated compounds were tested for cytotoxicity and antibacterial activities.
1874-3900/$ – see front matter ß 2013 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe. http://dx.doi.org/10.1016/j.phytol.2012.12.010
Please cite this article in press as: Lannang, A.M., et al., Ovalifolone A and B: New friedelane derivatives from Garcinia ovalifolia. Phytochem. Lett. (2013), http://dx.doi.org/10.1016/j.phytol.2012.12.010
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2
Fig. 2. Selected HMBC correlation of compounds 1 and 2.
Fig. 1. Structures of compounds 1 and 2.
27
2. Results and discussion
28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
The mixture of hexane and dichloromethane extract from the stem bark of G. ovalifolia was subjected to repeated column chromatography and 10 compounds were isolated: two new friedelane derivatives, ovalifolone A (1) and ovalifolone B (2); and eight known secondary metabolites, namely friedelin (3) (Ngouamegne et al., 2008), endodesmiadiol (4) (Ngouamegne et al., 2008), canophyllol (5) (Ngouamegne et al., 2008), canophyllal (6) (Ngouamegne et al., 2008), garcinane (7) (Meli Lannang et al., 2008), gallic acid (8) (Sudjaroen et al., 2012), isoxanthochimol (9) (Louh et al., 2008) and 3-methoxycheffouxanthone (10) (Meli Lannang et al., 2010). Compound 1 was obtained as a white amorphous solid. Its molecular formula was established as C30H48O3 from HREIMS data (m/z 456.35980 for [M]+), indicating seven degrees of unsaturation. The positive response to the Liebermann-Burchard test suggested the triterpenic nature of 1. The IR spectrum showed absorption bands characteristic of OH (3420 cm1) and carbonyl (1699 cm1) groups. Analysis of the 13C NMR (Table 1) in combination with
Table 1 NMR (1H: 500 MHz, Position
13
C: 125 MHz) data of compounds 1–2. Compound 1, CDCl3 1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 3-OH
DEPT and HMQC experiments revealed the presence of 30 signals including seven methyls (dc 32.8, 34.2, 19.0, 18.8, 18.8, 17.8 and 17.8), eleven sp3 methylenes (dc 38.3, 34.5, 34.4, 33.3, 32.3, 31.2, 31.1, 29.8, 29.0 and 17.9) among which one carbinol (dc 68.0), tree sp3 methines (dc 55.6, 52.0 and 39.4) and nine quaternary carbons (dc 142.5, 140.7, 39.5, 39.3, 36.7, 35.1 and 28.1) among which one carbonyl group (dc 194.9). The 1H NMR spectrum (Table 1) showed seven methyl singlets. These data were in agreement with the structure of the friedelane skeleton, ascribing the signal at dH 1.83 (s) to H-23 (Ngouamegne et al., 2008). This last signal showed a HMQC correlations with the shielded methyl carbon at dc 10.3 and HMBC correlation with the quaternary carbons at dc 140.7 (C-4), 142.5 (C-3) and the methyl carbon at dc 17.8 (C-24), establishing the ketone group in ring A at C-2 and the double bond between C-3 and C-4. This was supported by the chemical shifts at dH 2.53 (Ha1) and 2.42 (Hb-1) showing HMQC correlations with the methylene carbon at dc 32.3 and HMBC correlations (Fig. 2) with quaternary carbons at dc 194.9 (C-2), 142.5 (C-3), 39.5 (C-5) and the methine carbon at dc 55.6 (C-10). A primary OH group was evident in the 1H NMR spectrum by two doublets located at dH 3.64 d (J = 15.5 Hz)
2.53 2.42 – – – – 1.94 1.43 1.43 – 1.83 1.46 1.87 – – 1.41 1.31 – 1.29 1.40 – 1.33 1.87 1.83 1.11 1.08 0.95 1.10 3.64 3.65 0.95 0.99 –
Compound 2, CDCl3 13
H (1H, dd, J = 17.6, 3.9 Hz) (1H, dd, J = 17.6, 13.9 Hz)
(1H, m); 1.46 (1H, m) (1H, m), 1.56 (1H, m) (1H, m) (1H, m) (2H, m) (2H, m)
(2H, m) (2H, m) (1H, m) (2H, m) (2H, (2H, (3H, (3H, (3H, (3H, (3H, (1H, (1H, (3H, (3H,
m) m) s) s) s) s) s) d, J = 15.5 Hz) d, J = 15.5 Hz) s) s)
C
32.3 194.9 142.5 140.7 39.5 38.3 17.9 52.0 36.7 55.6 34.5 29.0 39.3 39.4 31.1 31.2 35.1 39.4 34.4 28.1 33.3 29.8 10.3 17.8 18.8 19.0 18.8 68.0 34.2 32.8 –
1
13
H
2.55 2.42 – 3.80 1.32 – 1.09 1.41 1.33 – 1.34 1.30 1.31 – – 1.41 1.86 – 1.29 1.28 – 1.22 1.02 1.05 1.07 0.91 0.93 1.08 3.68
(1H, dd, J = 3.5; 13.8 Hz) (1H, t, J = 13.8 Hz) (1H, dd, J = 3.8; 11.6 Hz) (1H, m) (1H, m); 1.86 (1H, m) (1H, m); 1.51 (1H, m) (1H, m) (1H, m) (2H, m) (2H, m)
(1H, m); 1.48 (1H, m) (2H, m) (1H, m) (2H, m) (2H, (1H, (3H, (3H, (3H, (3H, (3H, (2H,
m) m); 1.32 (1H, m) d, J = 6.0 Hz) s) s) s) s) br s)
0.99 (3H, s) 1.00 (3H, s) 3.56 (OH, d, J = 3.8 Hz)
C
36.1 211.8 76.8 54.5 38.0 40.5 17.5 52.5 37.6 60.3 34.5 31.2 38.1 38.0 31.3 29.9 35.1 39.4 34.4 28.1 31.2 33.3 10.8 14.2 17.6 19.0 19.1 68.0 34.3 31.2 –
Please cite this article in press as: Lannang, A.M., et al., Ovalifolone A and B: New friedelane derivatives from Garcinia ovalifolia. Phytochem. Lett. (2013), http://dx.doi.org/10.1016/j.phytol.2012.12.010
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Table 2 Determination of lethal dose LD50. Sample
10 mg/mL
5 mg/mL
2.5 mg/mL
1.25 mg/mL
0.02 mg/mL
LD50
7 Actinomycine D Berberine hydrochloride
100 100 n.m.
91 100 n.m.
45 100 n.m.
0 100 n.m.
n.m. 50 n.m.
2.69 mg/mL 0.02 mg/mL 26 mg/mL
Notes. Tests were carried out in duplicate. The mean percentage mortality was calculated for each concentration after the formula (A-N-B)Z1100, with A = number of dead larvas after 24 h; N = number of dead larvas before the sample was added B = number of dead larvas in the negative control after 24 h Z = total number of larvas. Mortality percentages were plotted against the logarithm of concentrations and LD50, at which 50% of the Artemia salina died, was determined from the graph. For Actinomycine D, LD50 Q3 directly resulted from the assay. The LD50 value of Berberine hydrochloride was taken from the literature (Saidnia et al., 2009). n.m. = not measured.
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98
and 3.65 (J = 15.5 Hz), which correspond to the carbinol methylene protons H-28. The presence of this group was supported by the signal of an oxygenated methylene carbon at dc 68.0 in the 13C NMR carbon spectrum. These carbinol protons showed long range correlations with the methine group at dc 39.4 (C-18), two methylene groups at dc 29.8 (C-22), 31.2 (C-16) and a quaternary carbon at dc 35.1 (C-17). The additional 1H and 13C NMR signals were similar to those reported for friedelane triterpene (Ngouamegne et al., 2008). Thus, compound 1 was established as 3, 28 dihydroxyfriedel-3-en-2-one (Fig. 1) and was trivially named ovalifolone A (Table 2). Compound 2 was isolated as colorless needles. Its IR spectrum exhibited bands of OH (3370 cm1) and carbonyl (1669 cm1) groups. The molecular formula was established as C30H50O3 from HREIMS data at (m/z 458.37520 for [M]+), indicating six degrees of insaturation. The positive response to the Liebermann-Burchard test suggested the triterpenic nature of compound 2. The 13C NMR spectrum (Table 1) showed 30 carbon signals, and the DEPT spectra indicated the presence of seven methyls, eleven methylenes including one carbinol (dc 68.0), five methines including one carbinol (dc 76.8), and seven quaternary carbons with one carbonyl at dc 211.8 ppm. The 1H NMR spectrum (Table 1) showed seven methyls signals, including six singlets (dH 1.08, 1.07, 1.00, 0.99, 0.93 and 0.91), and a doublet at dH 1.05 (J = 6.0 Hz), similar to those of endodesmiadol (Ngouamegne et al., 2008) The last signal was ascribed to H-23 since it showed a HMQC correlation with the shielded methyl carbon at dc 10.8 and crosspeaks HMBC with two methine carbon at dc 76.8 (C-3) and 54.5 (C-4), and with a quaternary carbon at dc 38.0 (C-5) establishing the hydroxyl group at C-3. The exchangeable proton (OH-3) resonated at dH 3.56 as a doublet (J = 3.8 Hz), showed long range correlations with the carbonyl group at dc 211.8 (C-2) and two methines at dc 76.8 (C-3) and (dc 54.5 (C-4) establishing a ketone group at C-2. The doublet of
Fig. 3. Selected NOESY and COSY of compound 2.
doublets at dH 3.85 (J = 3.8 and 11.6 Hz) corresponds to the proton 99 H-3. This proton was assigned to have b-orientation from the J 100 coupling constant and from NOESY experiments (Fig. 3), which 101 exhibited proximity to methyl C-23, methyl C-24 and H-1b. The 102 OH in position 3 was assigned unambiguously to have a- 103 orientation. The doublet of doublet at dH 2.55 (J = 13.8 and 104 3.2 Hz) and the broad triplet at dH 2.42 (J = 13.8 Hz) were assigned 105 to the methylene protons H-1a and H-1b, respectively. The 106 additional 1H and 13C NMR signals were similar to those of 107 friedelane (Ngouamegne et al., 2008). Thus, compound 2 was 108 established as 3a, 28 dihydroxyfriedelan-2-one (Fig. 1) and was 109 trivially named ovalifolone B. 110 Compounds 1, 2, 4, 5, 7 and 8 were tested for their biological 111 activity against various microbes by paper disk diffusion assay at 112 40 mg/paper disk, and against Artemia salina for cytotoxicity 113 assay at 10 mg/mL. Compound 7 responded positively in the 114 cytotoxicity assay and, the lethal dose LD50, which corresponds 115 to the concentration that killed 50% of the brine shrimps, was 116 determined to be 2.69 mg/mL. Other compounds did not show 117 LD50 values >10.0 mg/mL. Thus, compound 7 is more active than 118 to the well known anti cancer active alkaloid berberine 119 hydrochloride with LD50 = 26.0 mg/mL (Saidnia et al., 2012). Q2120 None of the tested samples showed activity against the bacteria 121 Staphylococcus aureus and Escherichia coli, the microfungi Candida 122 albicans and Mucor miehei and the plant pathogenic fungus 123 Rhizoctonia solani. Furthermore, compound 8 displayed moderate 124 activity against the plant pathogenic fungi Pythium ultimum 125 (14 mm). It should be noted that from a pharmacological point of 126 view, a good correlation between cytotoxic activities in the A. 127 salina test and antitumor, as well as antimalaria activities was 128 reported (Adoum, 2009). 129 3. Experimental
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3.1. General
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Optical rotations were measured in methanol solution on a JASCO digital polarimeter (model DIP-3600). The specific rotation is given in deg cm2 g1. IR spectra were recorded in CHCl3 on a JASCO A-302 IR spectrophotometer. The 1H, 13C, and 2D-NMR spectra were recorded on a Bruker AMX-500 spectrometer using CDCl3 as solvent. Homonuclear 1 H–1H connectivities were determined by using the COSY 458 experiment. One-bond 1 H–13C connectivities were determined by HMQC. Two- and three-bond 1 H–13C connectivities are determined by HMBC experiment. Proton chemical shifts are reported in d (ppm) with reference to the residual CDCl3 signal at d 7.26, and 13C NMR spectra are referenced to the central peak of CDCl3 at d 77.0. Coupling constants (J) were measured in Hz. The EIMS were recorded on a double-focusing mass spectrometer (Varian MAT 311A). HREIMS were recorded on a JEOL HX 110 mass spectrometer. Column chromatography was carried out with silica gel 60 (70–230 and 240–300 mesh sizes, E. Merck). Precoated silica gel TLC plates (E. Merck, F254) were used to
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check the purity of compounds, and ceric sulfate spray reagent was used for the visualization of compounds on TLC plates.
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3.2. Plant material
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The stem bark and leaves of Garcinia ovalifolia were collected at ‘‘Mont Kala’’ locality in Center Cameroon in April 2010 and identified by Mr. Nana Victor of the National Herbarium, Yaounde´, Cameroon, where a voucher specimen (ref. 55523/HNC) was deposited.
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3.3. Extraction and isolation
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The air-dried and powdered stem bark (4.5 kg) of Garcinia ovalifolia was separately extracted with MeOH at room temperature for 72 h. After evaporation under reduced pressure, 250.0 g of brown crude material were obtained. The crude extract (200 g) was suspended in aqueous MeOH (MeOH–H2O, 9:1, 2000 mL) and extracted with n-hexane (3 500 mL). The aqueous layer was then diluted with 60% MeOH and extracted with CH2Cl2 (3 500 mL), then with ethyl acetate. The extracts were concentrated under reduced pressure to afford 50.6, 60.8 and 25.0 g of n-hexane, dichloromethane and ethyl acetate extracts, respectively. The n-hexane and CH2Cl2 extracts were combined on the basis of TLC profile. This combined fraction was purified by column chromatography over silica gel 60 (230–400 mesh) and preparative TLC using a gradient system of n-hexane, CH2Cl2, ethyl acetate, and MeOH. All together, 210 subfractions (ca. 250 mL each) were collected and pooled on the basis of TLC analysis, leading to six main fractions (A–G). Fraction A (10.0 g) was the combination of subfractions 1–20, eluted with a mixture of n hexane–CH2Cl2 (8:2). Fraction B (10.0 g) was constituted of subfractions 21–49, eluted with a mixture of n -hexane–CH2Cl2 (6:4). Fraction C (10.0 g) was the combination of subfractions 50– 90, eluted with n -hexane–CH2Cl2 (1:1). Fraction D (10.0 g) was constituted of subfractions 91–122, eluted with pure CH2Cl2. Fraction E (10.0 g) was the combination of subfractions 123–152, eluted with CH2Cl 2–ethyl acetate (9:1). Fraction F (10.0 g) was constituted of subfractions 153–198, eluted with CH2Cl 2–ethyl acetate (8:2) and fraction G (9.0 g) constituted of subfraction 199–210, eluted with ethyl acetate–methanol (9:1). Fraction A was chromatographed over a silica gel 60C (20–40 mm) column with n -hexane–CH2Cl2 gradient. A total of 25 fractions of ca. 100 mL each were collected and combined on the basis of TLC profile. Subfractions 1–20 were further chromatographed on silica gel 60H (5–40 mm) with a mixture of n -hexane–CH2Cl2 (8:2) for elution to yield friedelan (3) (15.5 mg) and canophyllal (6) (8.5 mg). Fraction B was chromatographed over a silica gel 60C column with n -hexane–CH2Cl2 gradient. A total of 30 fractions of ca. 100 mL each were collected and combined on the basis of TLC. Subfractions 1–10 were further chromatographed over a silica gel 60H with a mixture of n -hexane–CH2Cl2 (4:1) to yield canophyllol (5) (10.0 mg), ovalifolone A (1) (12.0 mg), endodesmiadol (4) (7.5 mg), and gallic acid (8) (8.4 mg). Fraction C was chromatographed on a silica gel 60C column with n -hexane–CH2Cl2 gradient. All together, 35 fractions of ca. 100 mL each were collected and combined on the basis of TLC profile. Subfractions 21–35 were further chromatographed over a silica gel 60H with n hexane–CH2Cl2 (1:1) to yield ovalifolone B (2) (10.0 mg), garcinane (7) (6.0 mg), and 3-methylcheffouxanthone (10) (12.0 mg). Similarly, fraction G was chromatographed on a silica gel 60C column with n -hexane–CH2Cl2 (1:3) gradient. As a result, 20 fractions of ca. 100 mL each were collected and combined on the basis of TLC profile. Subfractions 8–15 were further chromatographed over silica gel 60H with n -hexane–CH2Cl2 (1:3) to yield isoxanthochimol (9) (40.0 mg).
3.4. Paper disk agar diffusion assay
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Agar test plates for bacteria and micro fungi are prepared as twolayer plates containing the microbial test strain in the uppermost layer (Maskey et al., 2002). Agar media are peptone agar for Bacillus subtilis and E. coli, Bacto nutrient agar for S. aureus, M2 agar for M. miehei and Sabouraud agar for C. albicans. Test plates for plant pathogen fungi R. solani, P. ultimum and Aphanomyces cochlioides are prepared as single layer plates of potato-dextrose agar (PDA). For bacteria and micro fungi, a few colonies of the test organism are picked with a wire loop from the original culture plate and introduced into a test tube containing 2 mL of fluid test medium. The tube is incubated for 12 h on a reciprocal shaker at 37 8C at 180 rpm. Petri dishes are filled with 25 mL of handwarm agar medium to give a depth of about 5 mm. The test strain suspension is 10-fold diluted with fluid test strain medium, mixed 1:4 with handwarm agar and 5 mL are poured on the first already stiff layer, to give a second 1 mm layer of so called ‘‘soft agar’’ due to less agar in this layer. For plant pathogen fungi, Petri dishes are filled with handwarm PDA agar to give a depth of about 5 mm and cooled down to become stiff. 1 cm 1 cm squares are cut from the growth front of well grown agar plates by a microbiologic hook and placed inverse onto the center of fresh plates. R. solani and P. ultimum plates are cultivated at 28 8C in the dark and A. cochlioides plates at room temperature (23 8C) in the dark until the diameter of the growing fungi measured around 6 cm. Above procedures are carried out under sterile conditions, i.e. media are autoclaved, agar plates and cultures are prepared in a clean bench, and wire loop and microbiologic hook are sterilized over the flame of a bunsen burner placed inside the clean bench. Six test samples and reference Cycloheximid are dissolved in DCM/MeOH 9:1, reference streptomycin sulfate in pure DMSO. 40 mL of a 1 mg/mL solution are applied on the disks. Solvents are evaporated for 1 h. Five paper disks impregnated with compound and one paper disk containing the corresponding reference are placed in a circle on the surface of each test agar plate with flamed forceps and gently pressed down to ensure contact. The plates containing bacteria and micro fungi are inverted and incubated for 12 h at 37 8C. Plates with plant pathogen fungi R. solani and P. ultimum are incubated for two to three days at 28 8C in the dark, and A. cochlioides at room temperature. The diameters of zones with complete inhibition of test strain growth are measured with a ruler and given in mm. The bacteria S. aureus, B. subtilis, E. coli and the microfungus C. albicans are Cameroonian clinical isolates; the microfungus M. miehei and the plant pathogen fungi originate from the Strain Collection Tu¨bingen of the Institute of Plant Pathology and Crop Protection, Go¨ttingen.
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3.5. Cytotoxicity assay
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The A. salina toxicity assay is a simple, rapid and inexpensive bench top assay and considered as a useful tool for preliminary assessment of general toxicity (Meyer et al., 1982). A separation funnel is filled with 1 L of filtered artificial seawater and 200 mg of A. salina eggs and kept under permanent light and at constant temperature until the larva hatch. 990 mL of seawater containing 20 or more larvas are pipetted in each well of a 24-well microtiter plate. Dead larvae are recorded under a microscope. 10 mL of 1 mg/mL solution of each compounds in DMSO is added to make an end concentration of 10 mg/mL. The tests are carried out in duplicate. For negative control, 10 mL of DMSO and for positive control, 10 mL of a 1 mg/mL solution of actinomycin D in DMSO is used. After incubation for 24 h at permanent light and constant temperature, dead larvas are counted. The plates are kept in the refrigerator for 24 hours to
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immobilize the larvae. The total number of larvas is calculated for each well. The mortality in % is determined with the formula (A–N– B)Z1100, with A = number of dead larvas after 24 h; N = number of dead larvas before the addition of tested compound; B = number of dead larvas in the negative control; Z = total number of larvas. Anhydrous A. salina eggs were purchased in a German pet shop.
Acknowledgments
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A.M.L. would like to thank the Alexander von HumboldtStiftung for providing Georg Forster Fellowship for experienced researchers. This article is also dedicated to the memory of Professor David Lontsi who passed away on December 22, 2008.
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3.6. Determination of LD50 values
References
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Lethal dose LD50 of compound 7 active at 10 mg/mL in the cytotoxicity assay against A. salina was determined. The sample was dissolved in DMSO and serial diluted to give end concentrations of 10, 5, 2.5, 1.25, 0.625 mg/mL. Tests were carried out in duplicate. For the positive control Actinomycine D, the dilution line was extended to the end concentration of 0.006 mg/mL, since the compound is extremely high toxic and a low value was expected. The mean percentage mortality was calculated for each concentration after the formula (A–N–B)Z1100 (see above). Mortality percentages were plotted against the logarithm of concentrations and LD50, at which 50% of the A. salina died, was determined from the graph. For the positive control Actinomycine D, LD50 directly resulted from the calculation of mortalities.
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3.7. Ovalifolone A (1)
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Amorphous powder, ½a25 D þ 1:8 ðc 0:03; acetoneÞ. IR (CHCl3): nmax 3450, 3100, 2943, 2360, 2009, 1733, 1699, 14921213, 1086 cm1. EI-MS: m/z 456 [M]+ (16), 425 (98), 426 (82), 289 (70), 137 (100). HREIMS: m/z 456.3598 (calc, 456.3603) for C30H48O3. 1H and 13C NMR data, see Table 1.
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Colorless powder, mp: 236–237 8C. ½a25 D þ 6:6 ðc 0:05; acetoneÞ. IR (CHCl3): nmax 3648, 3350, 2940, 2363, 2057, 1792, 1704, 1473 cm1. EI-MS: m/z 458 [M]+ (10), 427 (80), 289 (49), 137 (100). HREIMS: m/z 458.3752 (calc, 458.3760) for C30H50O3. 1H and 13C NMR data, see Table 1.
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