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New cytotoxic sesquiterpene lactones from Anthemis scrobicularis ab
a
Ahmed M. Zaghloul , Hasan S. Yusufoglu , Mohamad Ayman A. a
a
Salkini & Aftab Alam a
Pharmacognosy Department, College of Pharmacy, Salman Bin Abdulaziz University, Al-Kharj 11942, Kingdom of Saudi Arabia b
Pharmacognosy Department, College of Pharmacy, Mansoura University, Mansoura 35516, Egypt Published online: 17 Sep 2014.
To cite this article: Ahmed M. Zaghloul, Hasan S. Yusufoglu, Mohamad Ayman A. Salkini & Aftab Alam (2014) New cytotoxic sesquiterpene lactones from Anthemis scrobicularis, Journal of Asian Natural Products Research, 16:9, 922-929, DOI: 10.1080/10286020.2014.931377 To link to this article: http://dx.doi.org/10.1080/10286020.2014.931377
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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 9, 922–929, http://dx.doi.org/10.1080/10286020.2014.931377
New cytotoxic sesquiterpene lactones from Anthemis scrobicularis Ahmed M. Zaghloula,b*, Hasan S. Yusufoglua, Mohamad Ayman A. Salkinia and Aftab Alama a Pharmacognosy Department, College of Pharmacy, Salman Bin Abdulaziz University, Al-Kharj 11942, Kingdom of Saudi Arabia; bPharmacognosy Department, College of Pharmacy, Mansoura University, Mansoura 35516, Egypt
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(Received 27 January 2014; final version received 3 June 2014) Four new sesquiterpene lactones, 4a-hydroxy-guaia-10(14),11(13)-diene-12,6a-olide (1), 4a-hydroxy-9a-acetoxy-guaia-1(10),2-diene-12,6a-olide (4), 4a-hydroxy-9bacetoxy-guaia-1(10),2-diene-12,6a-olide (5), and 1a,4a-dihydroxy-9a-acetoxyguaia-10(14),2-diene-12,6a-olide (6), were isolated from the aerial parts of Anthemis scrobicularis. Their structures were elucidated on the basis of their IR, NMR, and MS spectroscopic data. In addition, two known sesquiterpene lactones micheliolide (2) and achillin (3) were also isolated. The cytotoxicity of some of the isolated compounds was tested against HCT 116, HepG-2, and MCF-7 cell lines. Micheliolide and 4a-hydroxyguaia-10(14),11(13)-diene-12,6a-olide showed pronounced inhibitory activity while 4a-hydroxy-9a-acetoxy-guaia-1(10),2-diene-12,6a-olide showed weak activity. Keywords: Anthemis scrobicularis; sesquiterpene lactone; cytotoxicity
1.
Introduction
Anthemis L., the second largest genus of the tribe Anthemideae, comprises nearly 210 species [1,2]. It is represented in Saudi Arabia by 19 species [3]. Anthemis scrobicularis Yavin, Fam. Asteraceae is an annual herb, growing in sand dunes and sandy areas, Arabian Peninsula, Jordan, and Palestine [3]. There is wide interest in research of the plants of Anthemis, especially their active components, because many of the plants are reported to have antifungal, antioxidant [4], antitumor, antiplasmodial, anthelminthic, schistosomicidal, cytotoxic, phytotoxic, and analgesic activities [5]. The genus Anthemis is reputed to contain sesquiterpene lactones [6–8]. Sesquiterpene lactones constitute a large group of biologically active plant chemicals that have been identified in several plant families [9,10]. However, the greatest numbers are found in the Asteraceae family with over 3000 reported
different structures [11,12]. They have been shown to exhibit a wide range of biological activities. Reviewing the current literature, nothing was traced concerning the chemical constituents and biological activities of A. scrobicularis. The present work reports the isolation, structure elucidation, and cytotoxic activity of the sesquiterpene lactone content of A. scrobicularis. 2. Results and discussion The aerial parts of A. scrobicularis were extracted with methanol, and the total sesquiterpene lactone content was prepared by precipitating the other constituents with lead acetate. Chromatographic separation of the extract using silica gel columns, and purification on chromatotron afforded six guianolides (1 – 6), which were characterized through their UV, IR, LC/MS, and 1H NMR, 13C NMR, DEPT, and 2D NMR spectral data (Figure 1).
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Journal of Asian Natural Products Research 14
923
O
H 2 3
10
9
6
7
1
8 5
4 HO H 15
O
HO
11 12
H
H O
O
13
1
2
3
O
O
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O
O
O
O
HO
O
O HO
HO H
O
HO
H
H
O
O
O O
4
O
O
5
6
Figure 1. Structures of the isolated compounds 1 – 6.
Compounds 2 and 3 were identified as micheliolide and achillin as their spectral data were in full agreement with those reported for micheliolide [13 – 15] and achillin [16 –19]. HRLCMS of compound 1 displayed the molecular ion peak at m/z 248.1416 [M]þ (calcd for C15H20O3, 248.1412). Its NMR spectral data were almost similar to compound 2 except the disappearance of the signals assigned to the methyl group (C-14) of compound 2 and the appearance of two signals at d 5.01 (1H, d, J ¼ 3.0 Hz) and 4.97 (1H, d, J ¼ 3.0 Hz) that could be assigned to an exomethylene group at position 14 (Table 1). The coupling constant J1,5 ¼ 10.5 Hz indicated a-configuration of H-1. Consequently, it was elucidated as 4a-hydroxy-guaia-10 (14),11(13)-diene-12,6a-olide (CHEMBL 507441). To the best of our knowledge, this compound has not been isolated before from natural sources, but has been synthesized and tested as anticancer agent [20].
The structure of compound 4 was established as 4a-hydroxy-9a-acetoxyguaia-1(10),2-diene-12,6a-olide based on its spectral data. HRLCMS displayed the molecular ion peak at m/z 306.1469 [M]þ (calcd for C17H22O5, 306.1467). IR spectrum showed absorption bands at 1765 (CvO lactone) and 1715 (CvO acetate) cm21 for lactone and acetate groups. 13C NMR and DEPT spectra displayed signals for 17 carbons (4 CH3, 1 CH2, 7 CH, and 5C). 1H– 1H COSY spectrum displayed the cross-peak of the proton doublet at d 3.03 (H-5) and the triplet at d 4.15 (H-6), which in turn has a correlation with the proton at d 2.75 (dt, H-7) and H-7 correlated with the protons at d 2.03 (ddd, H-8b), 1.59 (ddd, H8a), and 2.63 (dd, H-11). The later has correlation with the methyl at d 1.13 (d, H13). The two protons at d 2.03 (ddd, H-8b) and 1.59 (ddd, H-8a) have correlations with the downfield proton signal at d 5.37 (dd, H-9). The chemical shift, multiplicity, and lack of further correlation of the proton at d 5.37 (dd, H-9) suggested that C-9 is
924 Table 1.
A.M. Zaghloul et al. 1
H NMR spectral data of compounds 1, and 4 – 6 (500 MHz, CDCl3). 1
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5
6
– 6.25, d(5.8) 6.01, d (5.8)
– 6.44, d (5.5) 6.03, d (5.5)
– 5.82, d (6.0) 6.06, d (6.0)
3.03, d (10.8) 4.15, t (10.8) 2.75, dt (10.8, 10.8, 1.8) 2.03, ddd (14.0, 4.0, 1.8) 1.59, ddd (14.0, 10.8, 2.0) 5.37, dd (4.0, 2.0)
2.90– 2.86, m 4.38, t (10.9) 2.90– 2.86, m 2.19– 2.16, m 1.73– 1.69, m 5.41, br d (4.6)
2.05, d (10.2) 4.52, t (10.2) 2.41 – 2.35, m 2.41 – 2.35, m 1.79 – 1.75, m 5.74, m
2.63, dd (10.8, 7.7) 1.13, d (7.7)
2.69– 2.62, m 1.20, d, (7.5)
2.67 – 2.61, m 1.15, s
1.84, s
1.95, s
1.34, s – 2.01, s
1.57, s – 2.08, s
5.23, d (1.6) 4.92, d (1.6) 1.53, s – 2.08, s
d ppm, multiplicity (J, Hz)
Positions 1 2 3a 3b 5 6 7 8a 8b 9a 9b 11 13a 13b 14a 14b 15 CvO CH3
4
3.03, dd (10.5, 9, 2) 1.84 – 1.81, m 1.96 – 1.92, m 1.84 – 1.81, m 2.38, t (10.5) 4.04, t (10.5) 2.78 – 2.73, m 2.29 – 2.24, m 1.39 – 1.35, m 2.69 – 2.65, m 1.96 – 1.92, m – 6.24, d (3.0) 5.53, d (3.0) 5.01, d (3.0) 4.97, d (3.0) 1.32, s – –
oxygenated and a double bond between C-1 and C-10. The downfield proton doublets at d 6.25 (d, H-2) and 6.01 (d, H-3) are correlated indicating the double bond at C-2. HMBC spectrum displayed the correlation of the protons at d 1.13 (3H, d, H-13) and the carbon at d 179.1 (C-12) confirming a-methyl-g-lactone moiety. The chemical shift and the multiplicity of the proton at d 5.37 (dd, H-9) indicated that C-9 is oxygenated. Besides, its correlation with the methyl carbon at d 22.3 (C-14) and with the carbon at d 40.3 (C-7) and the carbon at d 170.7 (CvO of the acetate) indicated the placement of the acetate group at position 9. Figure 2 shows some important HMBC correlations of compound 4. The relative stereochemistry of compound 4 was established based on coupling constant consideration, NOESY experiment, and relative to the biologically rationalized a-configuration of H-7 and b-configuration of H-6 [21]. The relative configuration of H-5 (a), H-6 (b), and H-7 (a) was deduced from the coupling constants (J5,6 ¼ 10.8 and J6,7 ¼ 10.8 Hz), in agreement with the trans axial deposition
O
H
H H H HO
H
O
H H
H O
O
Figure 2. Some important HMBC correlations of compound 4.
of these protons. NOESY correlations between H3-15 and H-6 and between H313 and H-6 suggested b-configuration of the methyl groups. The absence of NOESY correlation between H-9 and H-7 as well as the presence of correlation between H-9 and H3-14 as well as the coupling constants (J7a,8a ¼ 10.8 Hz), (J7a,8b ¼ 1.8 Hz), (J8b,9a ¼ 2.0 Hz), and (J8a,9a ¼ 4.0 Hz) suggested the b-configuration of H-9. These results were further confirmed by
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Figure 3. Molecular confirmation of NOESY correlations of compounds 4 and 5.
comparing data, in parts, of the compound 4 with published data [22,23] of partially similar compounds. Figure 3 shows the molecular confirmation of NOESY correlations of compounds 4 and 5. Compound 5 gave spectroscopic data almost identical to those of compound 4 except for the chemical shifts of H-7, H-8a, H-8b and H-9. Its NOESY spectrum did not show such correlation between H-9 and H3-14 demonstrating its a-configuration. Its HRLCMS showed a molecular ion peak at m/z 306.1387 [M]þ. Accordingly, compound 5 is determined as 4a-hydroxy-9bacetoxy-guaia-1(10),2-diene-12,6a-olide. Table 2.
13
H NMR and 13C NMR spectral data of compound 6 were almost similar to those of compound 4 except for the absence of the signals assigned to the methyl group (C-14) of compound 4 and the appearance of two doublets at d 5.23 (1H, d, J ¼ 1.6 Hz) and 4.92 (1H, d, J ¼ 1.6 Hz) that could be assigned to an exomethylene group at position 14 (Table 2). Besides, the chemical shift of C-1 (d 85.2) indicated hydroxylation of position 1. Its HSQC, COSY, HMBC, and HRLCMS displayed the molecular ion peak at m/z 322.1412 [M]þ (calcd for C17H22O6, 322.1416) confirmed compound 6 as 1a,4a-dihydroxy-9aacetoxy-guaia-10(14),2-diene-12,6a-olide.
C NMR spectral data of the isolated compounds 1 and 4– 6 (125 MHz, CDCl3). 1
4
5
6
141.8 (C) 132.6 (CH) 141.2 (CH) 83.2 (C) 55.0 (CH) 81.7 (CH) 41.0 (CH) 28.6 (CH2) 74.5 (CH) 128.6 (C) 38.9 (CH) 178.9 (C) 10.0 (CH3) 22.2 (CH3) 21.2 (CH3) 170.5 (C) 29.5 (CH3)
85.2 (C) 134.0 (CH) 144.2 (CH) 82.8 (C) 67.3 (CH) 81.1 (CH) 42.2 (CH) 31.0 (CH2) 71.6 (CH) 150.5 (C) 39.1 (CH) 179.0 (C) 10.2 (CH3) 110.5 (CH2) 25.2 (CH3) 170.2 (C) 21.3 (CH3)
d (DEPT)
Carbon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 CvO CH3
1
43.9 (CH) 26.2 (CH2) 40.2 (CH2) 79.7 (C) 55.7 (CH) 84.1 (CH) 47.4 (CH) 31.5 (CH2) 39.3 (CH2) 148.1 (C) 138.7 (C) 169.9 (C) 120.8 (CH2) 112.7 (CH2) 23.9 (CH3) – –
140.3 (C) 130.3 (CH) 143.2 (CH) 83.8 (C) 58.9 (CH) 82.2 (CH) 40.3 (CH) 28.9 (CH2) 74.3 (CH) 127.2 (C) 38.9 (CH) 179.1 (C) 10.2 (CH3) 22.3 (CH3) 25.3 (CH3) 170.7 (C) 21.3 (CH3)
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A.M. Zaghloul et al. sesquiterpene lactones due to interaction with biological nucleophiles such as cysteine sulfhydryl groups of the target proteins [20]. Compound 4 showed very weak cytotoxic activity as it lacks the a-methylene g-lactone moiety required for the activity (Figure 4).
To the best of our knowledge, compounds 1, 4, 5, and 6 are new and have not been isolated before from any natural sources. The results in Tables 3 and 4 indicated that the isolated compounds 1 and 2 possess cytotoxic activity comparable to that of doxorubicin (Dox) against the three tested cell lines and compound 4 has a weak cytotoxic activity. These results are in accordance with the previous reports that the previously synthesized compound 1 (CHEMBL 507441) revealed cytotoxicity against human THP1 cells at 20 mM by (3(4,5-dimethylthiazo-2-yl)-2,5-diphenyl-tetrazolium bromide) MTT assay and inhibition of NF-kappaB-mediated transcriptional activity transfected in PMAstimulated HEK293 cells after 24 h by luciferase reporter gene assay.1 Compound 2 (micheliolide) was reported to reduce the propagation of acute myelogenous leukemia stem cells [14,20]. It is generally perceived that the a-methylene g-lactone moiety is responsible for the biological activity of
3. Experimental 3.1 General experimental procedures UV spectra were recorded on a JASCO V-630 UV – Vis Spectrophotometer (JASCO Co., Ltd, Tokyo, Japan). IR spectra were recorded on a JASCO FT/IR-4100 type A (JASCO Co., Ltd). MS data were obtained on an AQUITY Ultra Performance LC equipped with LCT Premier XE Micro MASS (Waters, Milford, MA, USA). NMR spectra were run on a Bruker VX500 NMR spectrometer (Fa¨llanden, Switzerland) operating at 500 and 125 MHz for 1H NMR and 13 C NMR, respectively. Isolation of some compounds was carried out using Harrison Research Chromatotron, model 7924T (TSquared Technology, Inc., Palo Alto, CA, USA) and silica gel 60 PF254 (4 mm) (Merck, Billerica, MA, USA).
Table 3. IC50 values of the isolated compounds 1, 2, and 4 against cancer cell lines.
3.2 Plant material The aerial parts of A. scrobicularis were collected from the sandy areas near AlKharj governorate, Saudi Arabia in April 2013. The plant was kindly authenticated by Dr Yousef Yaquob, Department of Pharmacognosy, College of
IC50 (mg/ml)
Compound 1 Compound 2 Compound 4 DOX
HCT 116
HepG-2
MCF-7
8.95 8.34 46.53 4.62
8.16 9.61 – 5.76
18.75 18.25 46.51 6.35
Table 4. Cytotoxicity of compounds 1, 2, and 4 against cancer cell lines at concentrations of 5, 12.5, 25, and 50 mg/ml in comparison with Dox. Surviving fraction HCT 116
HepG-2
MCF-7
Conc. (mg/ml)
Dox
1
2
4
Dox
1
2
4
Dox
1
2
4
0 5 12.5 25 50
1 0.452 0.278 0.244 0.237
1 0.512 0.411 0.367 0.332
1 0.553 0.423 0.302 0.301
1 0.824 0.734 0.654 0.476
1 0.331 0.211 0.188 0.162
1 0.421 0.334 0.298 0.256
1 0.456 0.313 0.286 0.246
1 0.811 0.752 0.634 0.564
1 0.511 0.432 0.394 0.326
1 0.579 0.519 0.481 0.413
1 0.671 0.536 0.456 0.385
1 0.847 0.647 0.587 0.477
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Journal of Asian Natural Products Research
Figure 4. Cytotoxicity of compounds 1, 2, and 4 against cancer cell lines at concentrations of 5, 12.5, 25, and 50 mg/ml in comparison with Dox.
Pharmacy, King Saud University, AlRiyadh, Kingdom of Saudi Arabia. A voucher specimen (SAU-CPH-12002) is maintained in the herbarium of College of Pharmacy, Salman Bin Abulaziz University. The plant material was air dried and reduced to fine powder.
3.3
Extraction and isolation
Powdered A. scrobicularis (2 kg) was extracted by percolation in methanol (3 £ 4 l) at room temperature for 3 days. The combined methanolic extract was concentrated in rotary evaporator at 458C to 500 ml and then diluted with distilled water
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A.M. Zaghloul et al.
(500 ml). Lead acetate solution was added dropwise until no more precipitate is formed and then filtered. The filtrate was extracted with chloroform (4 £ 200 ml) and concentrated in a rotary evaporator at 458C to afford a yellowish brown residue (20 g). The residue was applied to the top silica gel column and then gradiently eluted with chloroform-containing increasing proportions of ethyl acetate to afford five fractions. Each fraction was separately subjected to separation by repeated silica gel column using gradient elution with chloroform-containing increasing proportions of ethyl acetate. Further purification was performed using chromatotron and chloroform–ethyl acetate mixture as an eluting solvent. Six pure compounds were obtained and designated compounds 1–6. 3.3.1 Compound 1 Colorless gummy material; UV: lmax 251 nm; IR: nmax 3525 (OH), 2940 (CH), 1773 (CvO lactone), 1642 (CvC), 1618, 1263, 1205 (CZO lactone) cm21. For 1H NMR (500 MHz, CDCl3) spectral data, see Table 1, and for 13C NMR (125 MHz, CDCl 3) spectral data, see Table 2. HRLCMS: m/z 248.1416 [M]þ (calcd for C15H20O3, 248.1412). 3.3.2
Compound 4
White needle crystals; UV: lmax 245 nm, IR: nmax 3487 (OH), 2946 (CH), 1765 (CvO lactone), 1715 (CvO acetate), 1639 (CvC), 1615, 1260, 1210 (CZO lactone) cm21. For 1H NMR (500 MHz, CDCl3) spectral data, see Table 1, and for 13 C NMR (125 MHz, CDCl3) spectral data, see Table 2. HRLCMS: m/z 306.1469 [M]þ (calcd for C17H22O5, 306.1467). 3.3.3 Compound 5 Colorless gummy material; UV: lmax 248 nm, IR: nmax 3490 (OH), 2951 (CH), 1769 (CvO lactone), 1695 (CvO acet-
ate), 1640 (CvC), 1613, 1262, 1208 (CZO lactone) cm21. For 1H NMR (500 MHz, CDCl3) spectral data, see Table 1, and for 13C NMR (125 MHz, CDCl3) spectral data, see Table 2. HRLCMS: m/z 306.1387 [M]þ (calcd for C17H22O5, 306.1467). 3.3.4
Compound 6
Colorless gummy material; UV: lmax 238 nm, IR: nmax 3485 (OH), 2955 (CH), 1770 (CvO lactone), 1692 (CvO acetate), 1642 (CvC), 1617, 1263, 1215 (CZO lactone) cm21. For 1H NMR (500 MHz, CDCl3) spectral data, see Table 1, and for 13 C NMR (125 MHz, CDCl3) spectral data, see Table 2. HRLCMS: m/z 322.1412 [M]þ (calcd for C17H22O6, 322.1416). 3.4 Measurement of potential cytotoxicity by sulforhodamine B assay The cytotoxic activity of the isolated compounds was done by the sulforhodamine B assay [24] in the National Cancer Institute, Cairo, Egypt. The isolated compounds 1, 2, and 4 were screened for their in vitro cytotoxic activity using the colorimetric MTT assay against hepatocellular carcinoma (HepG-2), human colon cancer (HCT 116), and breast cancer (MCF-7) cell lines at different concentrations (5, 12.5, 25, and 50 mg/ml). Dox was used as a standard for comparative purposes. Stock solutions of the investigated compounds were prepared in dimethylsulfoxide. Potential cytotoxicity of the compounds was tested as follows: cells were plated in 96-multiwell plate (104 cells/well) for 24 h before treatment with the compound to allow attachment of cells to the wall of the plate. Different concentrations of the compounds under test (5, 12.5, 25, and 50 mg/ml) were added to the cell monolayer triplicate wells prepared for each individual dose. Monolayer cells were incubated with the compound for 48 h at 378C and in
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Journal of Asian Natural Products Research atmosphere of 5% CO2. After 48 h, cells were fixed with trichloroacetic acid, and washed and stained for 30 min with 0.4% w/v sulfo-rhodamine-B stain in 1% acetic acid. Unbound excess stain was removed by four washes with 1% acetic acid and then the protein-pound dye was extracted with 10 mM unbuffered Tris buffer (tris ( – hydroxymethyl)aminomethane). Color intensity was measured in an ELISA reader. The relation between surviving fraction and drug concentration is plotted to get the survival curve of each tumor cell line after the specified compound. Supplementary material 1
H NMR and 1C NMR, COSY, and DEPT of compound 4 are available as Supplementary material. Acknowledgement The authors extend their appreciation to the Deanship of Scientific Research at Salman Bin Abdulaziz University for the work through the project no. 48-M-1433.
Note 1.
http://pubchem.ncbi.nlm.nih.gov/summary/ summary.cgi?cid¼14466195&loc ¼ ec_rcs
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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
New cytotoxic sesquiterpene lactones from Anthemis scrobicularis ab
a
Ahmed M. Zaghloul , Hasan S. Yusufoglu , Mohamad Ayman A. a
a
Salkini & Aftab Alam a
Pharmacognosy Department, College of Pharmacy, Salman Bin Abdulaziz University, Al-Kharj 11942, Kingdom of Saudi Arabia b
Pharmacognosy Department, College of Pharmacy, Mansoura University, Mansoura 35516, Egypt Published online: 17 Sep 2014.
To cite this article: Ahmed M. Zaghloul, Hasan S. Yusufoglu, Mohamad Ayman A. Salkini & Aftab Alam (2014) New cytotoxic sesquiterpene lactones from Anthemis scrobicularis, Journal of Asian Natural Products Research, 16:9, 922-929, DOI: 10.1080/10286020.2014.931377 To link to this article: http://dx.doi.org/10.1080/10286020.2014.931377
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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 9, 922–929, http://dx.doi.org/10.1080/10286020.2014.931377
New cytotoxic sesquiterpene lactones from Anthemis scrobicularis Ahmed M. Zaghloula,b*, Hasan S. Yusufoglua, Mohamad Ayman A. Salkinia and Aftab Alama a Pharmacognosy Department, College of Pharmacy, Salman Bin Abdulaziz University, Al-Kharj 11942, Kingdom of Saudi Arabia; bPharmacognosy Department, College of Pharmacy, Mansoura University, Mansoura 35516, Egypt
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(Received 27 January 2014; final version received 3 June 2014) Four new sesquiterpene lactones, 4a-hydroxy-guaia-10(14),11(13)-diene-12,6a-olide (1), 4a-hydroxy-9a-acetoxy-guaia-1(10),2-diene-12,6a-olide (4), 4a-hydroxy-9bacetoxy-guaia-1(10),2-diene-12,6a-olide (5), and 1a,4a-dihydroxy-9a-acetoxyguaia-10(14),2-diene-12,6a-olide (6), were isolated from the aerial parts of Anthemis scrobicularis. Their structures were elucidated on the basis of their IR, NMR, and MS spectroscopic data. In addition, two known sesquiterpene lactones micheliolide (2) and achillin (3) were also isolated. The cytotoxicity of some of the isolated compounds was tested against HCT 116, HepG-2, and MCF-7 cell lines. Micheliolide and 4a-hydroxyguaia-10(14),11(13)-diene-12,6a-olide showed pronounced inhibitory activity while 4a-hydroxy-9a-acetoxy-guaia-1(10),2-diene-12,6a-olide showed weak activity. Keywords: Anthemis scrobicularis; sesquiterpene lactone; cytotoxicity
1.
Introduction
Anthemis L., the second largest genus of the tribe Anthemideae, comprises nearly 210 species [1,2]. It is represented in Saudi Arabia by 19 species [3]. Anthemis scrobicularis Yavin, Fam. Asteraceae is an annual herb, growing in sand dunes and sandy areas, Arabian Peninsula, Jordan, and Palestine [3]. There is wide interest in research of the plants of Anthemis, especially their active components, because many of the plants are reported to have antifungal, antioxidant [4], antitumor, antiplasmodial, anthelminthic, schistosomicidal, cytotoxic, phytotoxic, and analgesic activities [5]. The genus Anthemis is reputed to contain sesquiterpene lactones [6–8]. Sesquiterpene lactones constitute a large group of biologically active plant chemicals that have been identified in several plant families [9,10]. However, the greatest numbers are found in the Asteraceae family with over 3000 reported
different structures [11,12]. They have been shown to exhibit a wide range of biological activities. Reviewing the current literature, nothing was traced concerning the chemical constituents and biological activities of A. scrobicularis. The present work reports the isolation, structure elucidation, and cytotoxic activity of the sesquiterpene lactone content of A. scrobicularis. 2. Results and discussion The aerial parts of A. scrobicularis were extracted with methanol, and the total sesquiterpene lactone content was prepared by precipitating the other constituents with lead acetate. Chromatographic separation of the extract using silica gel columns, and purification on chromatotron afforded six guianolides (1 – 6), which were characterized through their UV, IR, LC/MS, and 1H NMR, 13C NMR, DEPT, and 2D NMR spectral data (Figure 1).
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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O
H 2 3
10
9
6
7
1
8 5
4 HO H 15
O
HO
11 12
H
H O
O
13
1
2
3
O
O
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O
O
O
O
HO
O
O HO
HO H
O
HO
H
H
O
O
O O
4
O
O
5
6
Figure 1. Structures of the isolated compounds 1 – 6.
Compounds 2 and 3 were identified as micheliolide and achillin as their spectral data were in full agreement with those reported for micheliolide [13 – 15] and achillin [16 –19]. HRLCMS of compound 1 displayed the molecular ion peak at m/z 248.1416 [M]þ (calcd for C15H20O3, 248.1412). Its NMR spectral data were almost similar to compound 2 except the disappearance of the signals assigned to the methyl group (C-14) of compound 2 and the appearance of two signals at d 5.01 (1H, d, J ¼ 3.0 Hz) and 4.97 (1H, d, J ¼ 3.0 Hz) that could be assigned to an exomethylene group at position 14 (Table 1). The coupling constant J1,5 ¼ 10.5 Hz indicated a-configuration of H-1. Consequently, it was elucidated as 4a-hydroxy-guaia-10 (14),11(13)-diene-12,6a-olide (CHEMBL 507441). To the best of our knowledge, this compound has not been isolated before from natural sources, but has been synthesized and tested as anticancer agent [20].
The structure of compound 4 was established as 4a-hydroxy-9a-acetoxyguaia-1(10),2-diene-12,6a-olide based on its spectral data. HRLCMS displayed the molecular ion peak at m/z 306.1469 [M]þ (calcd for C17H22O5, 306.1467). IR spectrum showed absorption bands at 1765 (CvO lactone) and 1715 (CvO acetate) cm21 for lactone and acetate groups. 13C NMR and DEPT spectra displayed signals for 17 carbons (4 CH3, 1 CH2, 7 CH, and 5C). 1H– 1H COSY spectrum displayed the cross-peak of the proton doublet at d 3.03 (H-5) and the triplet at d 4.15 (H-6), which in turn has a correlation with the proton at d 2.75 (dt, H-7) and H-7 correlated with the protons at d 2.03 (ddd, H-8b), 1.59 (ddd, H8a), and 2.63 (dd, H-11). The later has correlation with the methyl at d 1.13 (d, H13). The two protons at d 2.03 (ddd, H-8b) and 1.59 (ddd, H-8a) have correlations with the downfield proton signal at d 5.37 (dd, H-9). The chemical shift, multiplicity, and lack of further correlation of the proton at d 5.37 (dd, H-9) suggested that C-9 is
924 Table 1.
A.M. Zaghloul et al. 1
H NMR spectral data of compounds 1, and 4 – 6 (500 MHz, CDCl3). 1
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5
6
– 6.25, d(5.8) 6.01, d (5.8)
– 6.44, d (5.5) 6.03, d (5.5)
– 5.82, d (6.0) 6.06, d (6.0)
3.03, d (10.8) 4.15, t (10.8) 2.75, dt (10.8, 10.8, 1.8) 2.03, ddd (14.0, 4.0, 1.8) 1.59, ddd (14.0, 10.8, 2.0) 5.37, dd (4.0, 2.0)
2.90– 2.86, m 4.38, t (10.9) 2.90– 2.86, m 2.19– 2.16, m 1.73– 1.69, m 5.41, br d (4.6)
2.05, d (10.2) 4.52, t (10.2) 2.41 – 2.35, m 2.41 – 2.35, m 1.79 – 1.75, m 5.74, m
2.63, dd (10.8, 7.7) 1.13, d (7.7)
2.69– 2.62, m 1.20, d, (7.5)
2.67 – 2.61, m 1.15, s
1.84, s
1.95, s
1.34, s – 2.01, s
1.57, s – 2.08, s
5.23, d (1.6) 4.92, d (1.6) 1.53, s – 2.08, s
d ppm, multiplicity (J, Hz)
Positions 1 2 3a 3b 5 6 7 8a 8b 9a 9b 11 13a 13b 14a 14b 15 CvO CH3
4
3.03, dd (10.5, 9, 2) 1.84 – 1.81, m 1.96 – 1.92, m 1.84 – 1.81, m 2.38, t (10.5) 4.04, t (10.5) 2.78 – 2.73, m 2.29 – 2.24, m 1.39 – 1.35, m 2.69 – 2.65, m 1.96 – 1.92, m – 6.24, d (3.0) 5.53, d (3.0) 5.01, d (3.0) 4.97, d (3.0) 1.32, s – –
oxygenated and a double bond between C-1 and C-10. The downfield proton doublets at d 6.25 (d, H-2) and 6.01 (d, H-3) are correlated indicating the double bond at C-2. HMBC spectrum displayed the correlation of the protons at d 1.13 (3H, d, H-13) and the carbon at d 179.1 (C-12) confirming a-methyl-g-lactone moiety. The chemical shift and the multiplicity of the proton at d 5.37 (dd, H-9) indicated that C-9 is oxygenated. Besides, its correlation with the methyl carbon at d 22.3 (C-14) and with the carbon at d 40.3 (C-7) and the carbon at d 170.7 (CvO of the acetate) indicated the placement of the acetate group at position 9. Figure 2 shows some important HMBC correlations of compound 4. The relative stereochemistry of compound 4 was established based on coupling constant consideration, NOESY experiment, and relative to the biologically rationalized a-configuration of H-7 and b-configuration of H-6 [21]. The relative configuration of H-5 (a), H-6 (b), and H-7 (a) was deduced from the coupling constants (J5,6 ¼ 10.8 and J6,7 ¼ 10.8 Hz), in agreement with the trans axial deposition
O
H
H H H HO
H
O
H H
H O
O
Figure 2. Some important HMBC correlations of compound 4.
of these protons. NOESY correlations between H3-15 and H-6 and between H313 and H-6 suggested b-configuration of the methyl groups. The absence of NOESY correlation between H-9 and H-7 as well as the presence of correlation between H-9 and H3-14 as well as the coupling constants (J7a,8a ¼ 10.8 Hz), (J7a,8b ¼ 1.8 Hz), (J8b,9a ¼ 2.0 Hz), and (J8a,9a ¼ 4.0 Hz) suggested the b-configuration of H-9. These results were further confirmed by
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Figure 3. Molecular confirmation of NOESY correlations of compounds 4 and 5.
comparing data, in parts, of the compound 4 with published data [22,23] of partially similar compounds. Figure 3 shows the molecular confirmation of NOESY correlations of compounds 4 and 5. Compound 5 gave spectroscopic data almost identical to those of compound 4 except for the chemical shifts of H-7, H-8a, H-8b and H-9. Its NOESY spectrum did not show such correlation between H-9 and H3-14 demonstrating its a-configuration. Its HRLCMS showed a molecular ion peak at m/z 306.1387 [M]þ. Accordingly, compound 5 is determined as 4a-hydroxy-9bacetoxy-guaia-1(10),2-diene-12,6a-olide. Table 2.
13
H NMR and 13C NMR spectral data of compound 6 were almost similar to those of compound 4 except for the absence of the signals assigned to the methyl group (C-14) of compound 4 and the appearance of two doublets at d 5.23 (1H, d, J ¼ 1.6 Hz) and 4.92 (1H, d, J ¼ 1.6 Hz) that could be assigned to an exomethylene group at position 14 (Table 2). Besides, the chemical shift of C-1 (d 85.2) indicated hydroxylation of position 1. Its HSQC, COSY, HMBC, and HRLCMS displayed the molecular ion peak at m/z 322.1412 [M]þ (calcd for C17H22O6, 322.1416) confirmed compound 6 as 1a,4a-dihydroxy-9aacetoxy-guaia-10(14),2-diene-12,6a-olide.
C NMR spectral data of the isolated compounds 1 and 4– 6 (125 MHz, CDCl3). 1
4
5
6
141.8 (C) 132.6 (CH) 141.2 (CH) 83.2 (C) 55.0 (CH) 81.7 (CH) 41.0 (CH) 28.6 (CH2) 74.5 (CH) 128.6 (C) 38.9 (CH) 178.9 (C) 10.0 (CH3) 22.2 (CH3) 21.2 (CH3) 170.5 (C) 29.5 (CH3)
85.2 (C) 134.0 (CH) 144.2 (CH) 82.8 (C) 67.3 (CH) 81.1 (CH) 42.2 (CH) 31.0 (CH2) 71.6 (CH) 150.5 (C) 39.1 (CH) 179.0 (C) 10.2 (CH3) 110.5 (CH2) 25.2 (CH3) 170.2 (C) 21.3 (CH3)
d (DEPT)
Carbon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 CvO CH3
1
43.9 (CH) 26.2 (CH2) 40.2 (CH2) 79.7 (C) 55.7 (CH) 84.1 (CH) 47.4 (CH) 31.5 (CH2) 39.3 (CH2) 148.1 (C) 138.7 (C) 169.9 (C) 120.8 (CH2) 112.7 (CH2) 23.9 (CH3) – –
140.3 (C) 130.3 (CH) 143.2 (CH) 83.8 (C) 58.9 (CH) 82.2 (CH) 40.3 (CH) 28.9 (CH2) 74.3 (CH) 127.2 (C) 38.9 (CH) 179.1 (C) 10.2 (CH3) 22.3 (CH3) 25.3 (CH3) 170.7 (C) 21.3 (CH3)
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A.M. Zaghloul et al. sesquiterpene lactones due to interaction with biological nucleophiles such as cysteine sulfhydryl groups of the target proteins [20]. Compound 4 showed very weak cytotoxic activity as it lacks the a-methylene g-lactone moiety required for the activity (Figure 4).
To the best of our knowledge, compounds 1, 4, 5, and 6 are new and have not been isolated before from any natural sources. The results in Tables 3 and 4 indicated that the isolated compounds 1 and 2 possess cytotoxic activity comparable to that of doxorubicin (Dox) against the three tested cell lines and compound 4 has a weak cytotoxic activity. These results are in accordance with the previous reports that the previously synthesized compound 1 (CHEMBL 507441) revealed cytotoxicity against human THP1 cells at 20 mM by (3(4,5-dimethylthiazo-2-yl)-2,5-diphenyl-tetrazolium bromide) MTT assay and inhibition of NF-kappaB-mediated transcriptional activity transfected in PMAstimulated HEK293 cells after 24 h by luciferase reporter gene assay.1 Compound 2 (micheliolide) was reported to reduce the propagation of acute myelogenous leukemia stem cells [14,20]. It is generally perceived that the a-methylene g-lactone moiety is responsible for the biological activity of
3. Experimental 3.1 General experimental procedures UV spectra were recorded on a JASCO V-630 UV – Vis Spectrophotometer (JASCO Co., Ltd, Tokyo, Japan). IR spectra were recorded on a JASCO FT/IR-4100 type A (JASCO Co., Ltd). MS data were obtained on an AQUITY Ultra Performance LC equipped with LCT Premier XE Micro MASS (Waters, Milford, MA, USA). NMR spectra were run on a Bruker VX500 NMR spectrometer (Fa¨llanden, Switzerland) operating at 500 and 125 MHz for 1H NMR and 13 C NMR, respectively. Isolation of some compounds was carried out using Harrison Research Chromatotron, model 7924T (TSquared Technology, Inc., Palo Alto, CA, USA) and silica gel 60 PF254 (4 mm) (Merck, Billerica, MA, USA).
Table 3. IC50 values of the isolated compounds 1, 2, and 4 against cancer cell lines.
3.2 Plant material The aerial parts of A. scrobicularis were collected from the sandy areas near AlKharj governorate, Saudi Arabia in April 2013. The plant was kindly authenticated by Dr Yousef Yaquob, Department of Pharmacognosy, College of
IC50 (mg/ml)
Compound 1 Compound 2 Compound 4 DOX
HCT 116
HepG-2
MCF-7
8.95 8.34 46.53 4.62
8.16 9.61 – 5.76
18.75 18.25 46.51 6.35
Table 4. Cytotoxicity of compounds 1, 2, and 4 against cancer cell lines at concentrations of 5, 12.5, 25, and 50 mg/ml in comparison with Dox. Surviving fraction HCT 116
HepG-2
MCF-7
Conc. (mg/ml)
Dox
1
2
4
Dox
1
2
4
Dox
1
2
4
0 5 12.5 25 50
1 0.452 0.278 0.244 0.237
1 0.512 0.411 0.367 0.332
1 0.553 0.423 0.302 0.301
1 0.824 0.734 0.654 0.476
1 0.331 0.211 0.188 0.162
1 0.421 0.334 0.298 0.256
1 0.456 0.313 0.286 0.246
1 0.811 0.752 0.634 0.564
1 0.511 0.432 0.394 0.326
1 0.579 0.519 0.481 0.413
1 0.671 0.536 0.456 0.385
1 0.847 0.647 0.587 0.477
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Figure 4. Cytotoxicity of compounds 1, 2, and 4 against cancer cell lines at concentrations of 5, 12.5, 25, and 50 mg/ml in comparison with Dox.
Pharmacy, King Saud University, AlRiyadh, Kingdom of Saudi Arabia. A voucher specimen (SAU-CPH-12002) is maintained in the herbarium of College of Pharmacy, Salman Bin Abulaziz University. The plant material was air dried and reduced to fine powder.
3.3
Extraction and isolation
Powdered A. scrobicularis (2 kg) was extracted by percolation in methanol (3 £ 4 l) at room temperature for 3 days. The combined methanolic extract was concentrated in rotary evaporator at 458C to 500 ml and then diluted with distilled water
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A.M. Zaghloul et al.
(500 ml). Lead acetate solution was added dropwise until no more precipitate is formed and then filtered. The filtrate was extracted with chloroform (4 £ 200 ml) and concentrated in a rotary evaporator at 458C to afford a yellowish brown residue (20 g). The residue was applied to the top silica gel column and then gradiently eluted with chloroform-containing increasing proportions of ethyl acetate to afford five fractions. Each fraction was separately subjected to separation by repeated silica gel column using gradient elution with chloroform-containing increasing proportions of ethyl acetate. Further purification was performed using chromatotron and chloroform–ethyl acetate mixture as an eluting solvent. Six pure compounds were obtained and designated compounds 1–6. 3.3.1 Compound 1 Colorless gummy material; UV: lmax 251 nm; IR: nmax 3525 (OH), 2940 (CH), 1773 (CvO lactone), 1642 (CvC), 1618, 1263, 1205 (CZO lactone) cm21. For 1H NMR (500 MHz, CDCl3) spectral data, see Table 1, and for 13C NMR (125 MHz, CDCl 3) spectral data, see Table 2. HRLCMS: m/z 248.1416 [M]þ (calcd for C15H20O3, 248.1412). 3.3.2
Compound 4
White needle crystals; UV: lmax 245 nm, IR: nmax 3487 (OH), 2946 (CH), 1765 (CvO lactone), 1715 (CvO acetate), 1639 (CvC), 1615, 1260, 1210 (CZO lactone) cm21. For 1H NMR (500 MHz, CDCl3) spectral data, see Table 1, and for 13 C NMR (125 MHz, CDCl3) spectral data, see Table 2. HRLCMS: m/z 306.1469 [M]þ (calcd for C17H22O5, 306.1467). 3.3.3 Compound 5 Colorless gummy material; UV: lmax 248 nm, IR: nmax 3490 (OH), 2951 (CH), 1769 (CvO lactone), 1695 (CvO acet-
ate), 1640 (CvC), 1613, 1262, 1208 (CZO lactone) cm21. For 1H NMR (500 MHz, CDCl3) spectral data, see Table 1, and for 13C NMR (125 MHz, CDCl3) spectral data, see Table 2. HRLCMS: m/z 306.1387 [M]þ (calcd for C17H22O5, 306.1467). 3.3.4
Compound 6
Colorless gummy material; UV: lmax 238 nm, IR: nmax 3485 (OH), 2955 (CH), 1770 (CvO lactone), 1692 (CvO acetate), 1642 (CvC), 1617, 1263, 1215 (CZO lactone) cm21. For 1H NMR (500 MHz, CDCl3) spectral data, see Table 1, and for 13 C NMR (125 MHz, CDCl3) spectral data, see Table 2. HRLCMS: m/z 322.1412 [M]þ (calcd for C17H22O6, 322.1416). 3.4 Measurement of potential cytotoxicity by sulforhodamine B assay The cytotoxic activity of the isolated compounds was done by the sulforhodamine B assay [24] in the National Cancer Institute, Cairo, Egypt. The isolated compounds 1, 2, and 4 were screened for their in vitro cytotoxic activity using the colorimetric MTT assay against hepatocellular carcinoma (HepG-2), human colon cancer (HCT 116), and breast cancer (MCF-7) cell lines at different concentrations (5, 12.5, 25, and 50 mg/ml). Dox was used as a standard for comparative purposes. Stock solutions of the investigated compounds were prepared in dimethylsulfoxide. Potential cytotoxicity of the compounds was tested as follows: cells were plated in 96-multiwell plate (104 cells/well) for 24 h before treatment with the compound to allow attachment of cells to the wall of the plate. Different concentrations of the compounds under test (5, 12.5, 25, and 50 mg/ml) were added to the cell monolayer triplicate wells prepared for each individual dose. Monolayer cells were incubated with the compound for 48 h at 378C and in
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Journal of Asian Natural Products Research atmosphere of 5% CO2. After 48 h, cells were fixed with trichloroacetic acid, and washed and stained for 30 min with 0.4% w/v sulfo-rhodamine-B stain in 1% acetic acid. Unbound excess stain was removed by four washes with 1% acetic acid and then the protein-pound dye was extracted with 10 mM unbuffered Tris buffer (tris ( – hydroxymethyl)aminomethane). Color intensity was measured in an ELISA reader. The relation between surviving fraction and drug concentration is plotted to get the survival curve of each tumor cell line after the specified compound. Supplementary material 1
H NMR and 1C NMR, COSY, and DEPT of compound 4 are available as Supplementary material. Acknowledgement The authors extend their appreciation to the Deanship of Scientific Research at Salman Bin Abdulaziz University for the work through the project no. 48-M-1433.
Note 1.
http://pubchem.ncbi.nlm.nih.gov/summary/ summary.cgi?cid¼14466195&loc ¼ ec_rcs
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