|Year : 2020 | Volume
| Issue : 70 | Page : 391-395
Cytotoxic isoprenoids from Xanthium strumarium linn.
Hajer S Alorfi1, Amani A Alshehry1, Mohamed A Ghandourah2, Nahed O Bawakid1, Mahmoud A Elfaky3, Aasim M Ali4, Walied M Alarif2
1 Department of Chemistry, Faculty of Sciences, King Abdulaziz University, Jeddah, Saudi Arabia
2 Department of Marine Chemistry, Faculty of Marine Sciences, King Abdulaziz University, Jeddah, Saudi Arabia
3 Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
4 Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, 1433 Ås, Norway
|Date of Submission||03-Jan-2020|
|Date of Decision||12-Feb-2020|
|Date of Acceptance||17-Mar-2020|
|Date of Web Publication||28-Aug-2020|
Walied M Alarif
Department of Marine Chemistry, Faculty of Marine Sciences, King Abdulaziz University, PO. Box 80207, Jeddah 21589
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background and Objective: Xanthium strumarium is a widespread medicinal plant species; particularly fruits and roots are known for improving memory, voice, and appetite as well as curing of poisonous bites of insects and epilepsy. Materials and Methods: The aerial parts of X. strumarium were extracted with a combination of organic solvents. The exhaustively dried organic extract was fractionated until obtaining pure individuals by employing the appropriate chromatographic techniques. The spectral information obtained from different nuclear magnetic resonance experiments, mass, infrared, and ultraviolet spectra were the keys to elucidate the chemical structures. Results: Nine compounds (1-9) were obtained: a germacrane sesquiterpene (1), five xanthatin-type sesquiterpenoids (5-9) with α-methylene-γ-lactone moiety, including the new one, methoxy xanthanol (9), a benzopyran derivative not previously found in nature 3,4-diepoxy-2,2-dimethyl-2H-1-benzopyran-6-carboxaldehyde (2), and coumarin (3), along with the C-28 steroid campesterol (4). Conclusion: Most of the compounds under the study showed an appreciated cytotoxic activity against HCT116 and HepG2 cancer cell lines.
Keywords: Asteraceae , cytotoxicity, medicinal plants, Spectroscopy , terpenoids
|How to cite this article:|
Alorfi HS, Alshehry AA, Ghandourah MA, Bawakid NO, Elfaky MA, Ali AM, Alarif WM. Cytotoxic isoprenoids from Xanthium strumarium linn. Phcog Mag 2020;16:391-5
|How to cite this URL:|
Alorfi HS, Alshehry AA, Ghandourah MA, Bawakid NO, Elfaky MA, Ali AM, Alarif WM. Cytotoxic isoprenoids from Xanthium strumarium linn. Phcog Mag [serial online] 2020 [cited 2020 Oct 28];16:391-5. Available from: http://www.phcog.com/text.asp?2020/16/70/391/293787
- A new methoxy xanthanol and a new benzopyran derivative in addition to seven known isoprenoids compounds were isolated from Xanthium strumarium Linn .
- The biological investigation of most of isolated compounds presented significant activity against HCT116 and HepG2 cancer cell lines.
Abbreviations used: NMR: Nuclear magnetic resonance; MS: Mass spectrometry; UV: Ultraviolet spectroscopy; IR: Infrared radiation; EIMS: Electron ionization mass spectra; TLC: Thin-layer chromatography; PTLC: Preparative thin-layer chromatography; HSQC: Heteronuclear single-quantum correlation; COSY: Correlated spectroscopy; HMBC: Heteronuclear multiple-bond correlation.
| Introduction|| |
The genus Xanthium, with 25 members, is a small genus belonging to the large plant family Asteraceae .,Its species are distributed in almost all continents., They were used in the Traditional Chinese Medicine for the treatment of ulcer, arthritis, pruritus, cancer, herpes, and nasal sinusitis.,,
Xanthium strumarium Linn. (Cocklebur) is a herbaceous daisy plant, originated in tropical America and widely distributed in the world. Literature screening of the previous studies focused on the medicinal usage and chemical constituents of X. strumarium revealed the broadness appearance in folk medicine as prophylactic, diuretic, sudorific, sialogogue, sedative agent, and a remedy for malaria. It is noteworthy to mention that xantholide, a main constituent sesquiterpene lactone in X. strumarium , displayed a high potency against chloroquine-resistant Plasmodium falciparum . Polyphenols, plastoquinone, and tocopherols are the main constituents of the fruits of X. strumarium , whereas the aerial parts showed to contain daucane- and xanthane-type sesquiterpenoids.,
In pharmaceutical industry, the combinatorial chemistry has attracted the attention to the synthetic rather than the natural compounds due to high-throughput and large-scale applications. Nevertheless, natural compounds are more diverse and supply higher structural complexity than the synthetic equivalents. Moreover, in drug-like enrichment, natural metabolites are much higher than the synthesized counterparts.
Colorectal cancer is already the third leading cause of cancer death in the world and its incidence is steadily rising in developing nations, while liver cancer is the fifth most common cancer, accounting for 9.1% of all cancer deaths worldwide This manuscript is interested in the isolation and evaluation of the cytotoxicity of secondary metabolites from the indigenous medicinal plant X. strumarium , known in the Arabian area as “Losiq.” It was collected from River Nile State, Sudan. Its organic extract afforded nine compounds 1–9: six sesquiterpenoids 1 and 5–9, a new natural benzopyran derivative (2) previously known as synthetic product, and coumarin (3) along with the C-28 steroid, campesterol (4). All compounds were tested against two cancer cell lines HCT116 (colon cancer) and HepG2 (liver cancer) using sulforhodamine B assay. Most of the compounds under the study showed an appreciated cytotoxic activity against HCT116 and HepG2 cancer cell lines.
| Materials and Methods|| |
The materials and methods employed in this article are detailed elsewhere.
X. strumarium was collected from River Nile State, Sudan, in March 2017, and was identified by Prof. Jamal Sabar, Department of Biological Sciences, Faculty of Sciences, King Abdulaziz University (KAU), Jeddah, Saudi Arabia. A voucher specimen of X. strumarium has been deposited at the Department of Biological Sciences, Faculty of Sciences, KAU.
Extraction and isolation
The partially air-dried aerial parts (371.0 g) were soaked in a mixture of equal volumes of petroleum ether, methylene chloride, and methanol (4.5 L, two times at room temperature). The extract was then concentrated and successively portioned between n -hexane/water and chloroform/water. The chloroform extract (12.42 g) was chromatographed on an aluminum oxide column (750 g, 75 cm × 2.5 cm) and eluted successively with petroleum ether, petroleum ether–ether, and petroleum ether–ethyl acetate. 50 mL fractions were collected, thin-layer chromatography (TLC) was carried out for all fractions, and p -anisaldehyde-sulfuric acid and methanol-sulfuric acid reagents were employed for visualization. The fraction eluted with petroleum ether was further purified by preparative thin-layer chromatography (PTLC) (using petroleum ether as eluent); the violet band (R f0.76) appeared up spraying with p -anisaldehyde-sulfuric acid was collected to give an oily material (1, 1.7 mg). The fraction eluted with 25% diethyl ether in petroleum ether was further purified by PTLC (15% diethyl ether in petroleum ether) to give three bands; the purple band (R f 0.56) appeared up on spraying with p -anisaldehyde-sulfuric acid was collected to give an oily material (2, 0.5 mg). The violet band (R f 0.29) was collected as colorless solid (3, 1.7 mg). The dark purple band (R f 0.22) was collected as white solid material (4, 1.4 mg). The fraction eluted with 50% diethyl ether in petroleum ether was further purified by PTLC (50% diethyl ether in petroleum ether) to give a yellowish band (R f 0.35) which appeared up spraying with p -anisaldehyde-sulfuric acid was collected to give an oily material (5, 2.5 mg). The fraction eluted with 30% ethylacetate in n -hexane was further purified by PTLC (30% ethylacetate in n -hexane) to give a violet band (Rf 0.42) which appeared up spraying with p -anisaldehyde-sulfuric acid was collected to give an oily material (6, 1.8 mg). The fraction eluted with 40% ethylacetate in n -hexane was further purified by PTLC (50% ethyl acetate in n -hexane) to give three bands: a dark violet band (Rf 0.56) appeared up spraying with p -anisaldehyde-sulfuric acid was collected to give an oily material (7, 3.5 mg), a reddish band at R f0.41 was collected to give an oily material (9, 1.9 mg), and a light violet band at R f0.22 was collected to give an oily material (8, 2.0 mg).
Characterization of the isolated compounds
3,4 diepoxy-2,2-dimethyl-2H-1-benzopyran-6- carboxaldehyde (2)
Characterization of the 3,4 diepoxy-2,2-dimethyl-2H-1-benzopyran-6- carboxaldehyde (2) included pale yellow oil (0.5 mg); ultraviolet spectroscopy (UV) λmax(MeOH) 221, 253 nm; IR ύmax2925 (CH, st), 1705 (C=O, st), 3054 (=CH, st), and 1680 (C=C, Ar, st), 1497 cm−1; HR-ESI-MS m /z 204.0780 [M]+ (Calcd for C12H12O3, 204.0786);1 HNMR (Bruker WM 850 MHz); and13 C Nuclear magnetic resonance (NMR) (212.5 MHz) in CDCl3[Table 1].,
|Table 1:1H and13C Nuclear magnetic resonance spectral data for compounds 2 and 9|
Click here to view
Methoxy xanthanol (9)
Characterization of the methoxy xanthanol (9) included colorless oil (2.0 mg); UV λmax(MeOH) 218 nm; IR ύmax1375(gem -dimethyl, st) 1249(C-O), 1736 (C = O), 1767 (lactone), and 2922(CH) cm−1, HR-ESI-MS m/z 322.1774 [M]+(Calcd for C18H26O5, 322.1780);1 HNMR (Bruker WM 850 MHz); and13 C NMR (212.5 MHz) in CDCl3[Table 1].
Known natural compounds
The further isolated natural metabolites were identified as 1,5-dimethyl-8-(1-methylethylidene)-1,4-cyclodecadiene (1), coumarin (3), campesterol (4), tomentosin (5), 1'-hydroxytomentosin (6), xanthatin (7), and xanthanol (8) after comparison of their spectral and physical properties with the published data.,,,,,,
In vitro cytotoxic activity
The detailed biological method was mentioned previously by Skehan et al .,
| Results and Discussion|| |
Nine metabolites (1-9) were the net result of chromatographic separation of the organic extract of X. strumarium , out of them, two new natural compounds were identified: 2 (0.5 mg, 0.0001% yield), previously known as a synthetic product,, and 9 (2 mg, 0.0005% yield).
Compound 2 was isolated as pale yellow oil with specific rotation [α]D-59 (c 0.005, CHCl3). Its molecular formula was established as C12H12O3, derived from its HR-ESI-MS. The infrared radiation (IR) absorption spectra revealed characteristic bands at 1705, 1497, and 1361 cm−1 corresponding to aldehyde aromatic ring and gem -dimethyl functions.13 C NMR spectrum confirmed the presence of aldehyde carbon signal resonating at δC194.3 ppm, six signals in the aromatic region at δC155.0, 131.3, 130.4, 128.0, 120.0, and 117.4 ppm, three further oxygenated carbons resonating at δC79.3, 66.4, and 58.3 ppm, and two upfield signals at δC26.8 and 19.4 ppm.1 H NMR spectrum assigned the presence of two tertiary methyls at δH 1.26 and 0.95 ppm, two O-CH at δH 4.74 (d, J = 8.5 Hz) and 3.85 (d, J = 8.5 Hz) ppm, and three aromatic protons at 7.56 (br s), 7.70 (d, 8.5 Hz), and 7.22 (d, 8.5 Hz). Heteronuclear single-quantum correlation (HSQC)spectrum assigned all protons to their carbon atoms. The multiplicity pattern with the calculated coupling constants (J ) indicated the presence of 1, 2, 4-trisubstituted benzene ring. The molecular formula indicated that 2 has seven unsaturation sites, and the aldehyde function and the benzene ring accounted for five sites; therefore, 2 is a tricyclic compound. The presence of the oxirane ring was evidenced from the characteristic signals at δH/δC 3.85/66.4 and 4.74/58.3 ppm. The1 H-1 H correlated spectroscopy (COSY) displayed two proton sequences, H-3/H-4 and H-8/H-7, which support the previous assumptions. Heteronuclear multiple-bond correlation (HMBC) spectrum indicated the presence of the pyran ring through correlations from H-4 to C-5, 2, 10, methyl protons (0.95 ppm) to C-3,2 and the other methyl carbon, together with the13 C NMR signal at 155.0 ppm assigned to oxygenated aromatic carbon. Extensive interpretation of the HMBC results established the location of the aldehyde function through the correlations between aldehyde proton and C-6 together with that between H-5 and C-6 and the aldehyde carbon, as well as these two carbons with H-7. Compound 2 was previously identified as synthetic product., It is a new natural product named 3,4-diepoxy-2,2-dimethyl-2H-1-benzopyran-6-carboxaldehyde.
Compound 9 was isolated as gummy material. Structure elucidation of compound 9 commenced after developing of a characteristic color of terpenes on spraying with p -anisaldehyde reagent. The IR absorption spectrum of 9 evidenced the presence of α,β–unsaturated-γ-lactone moiety (1767 cm−1) which was validated by the UV absorption peak at 218 nm, in addition to acetyl group (1736 cm − 1). The molecular formula of 9 was established as C18H26O5 from the HR-ESI-MS and requires six unsaturation sites. The13 C NMR spectrum displayed 18 signals, two of which were ascribable to acetyl function (δC: 171.7 and 21.3) and one to methoxy carbon (δC: 50.9). The remaining 15 carbon atoms were categorized with the aid of distortionless enhancement by polarization transfer experiment into three CH-O functions (δC82.4, 74.4, and 68.2), two C-CH(C)-C (δC48.3 and 29.0), four CH2 groups (δC118.5, 42.8, 36.0, and 25.2), two methyl groups (δC20.1 and 19.6), one CH=(δC123.2) along with three quaternary carbons including two all-carbon linked quaternary (δC149.5 and 139.5), and one lactonized ketone group (δC170.1) [Table 1]. In the1 H NMR and HSQC spectra of 9
, in addition to two secondary methyls attached to saturated carbons (δH 1.28, d, J = 6.8 Hz and 1.18, d, J = 6.8 Hz), the δH value of the proton resonating at 4.31 (δH4.31, ddd, J = 12.8, 5.1, and 4.3 Hz) suggested that the OH function at carbon (δC 82.4) was involved in a lactone ring. The presence of α,β-unsaturated-γ-lactone moiety was substantiated by the signals due to exocyclic methylene resonating at δH6.16 and 5.44 with common J = 3.4 Hz. Based on the presence of two carbonyl functions, a trisubstituted double bond (δH 5.83, dd, J = 9.4 and 3.4 Hz), and a lactone ring, therefore, the compound 9 should be bicyclic.1 H-1 H COSY spectrum established the presence of two proton sequences: H-2/H-3, H-3/H-4, and H-4/H-15, this was first, the second one was correlations observed as H-7/H-6, H-7/H-8, H-6/H-5, H-10/H-9, H-10/H-14, and H-9/H-8. HMBC spectrum showed correlations that established the presence of a seven-membered ring (H-5 at δH5.83 with C-6, 1, 10 and H-9 at 4.31 with C-11, 6, 10), the location of the lactone ring (H-13 at 6.16 with C-12, 11, 7), and the location of the butyl side chain. The previous spectral data are most likely similar to xanthatin (7) metabolites frequently isolated from the plants of the genus Xanthium .,,,,,, The positions of the methoxy and acetoxy functions were assigned to C-2 and C-4, respectively, based on the chemical shift values, correlations observed in HMBC, and comparison with data from the literature.,,,,,, The name methoxy xanthanol was given to 9[Figure 1].
The cytotoxic effect of the isolated compounds was evaluated against two cancer cell lines HCT116 and HepG2. Based on the cytotoxicity criteria of pure compounds (IC50<4 μg/mL or <10 μM), the compounds were considered to be highly cytotoxic., Compounds 3 and 4 showed significant cytotoxic activities against HCT116 cells with the mean IC50 values of 0.016 ± 0.005 and 0.246 ± 0.021 μM, respectively, while compounds 1,7-9 showed moderate cytotoxic activities against HCT116 cells with the mean IC50 values of 19.576 ± 1.16, 14.392 ± 1.32, 24.165 ± 2.81, and 25.834 ± 2.03 μM, respectively. Compounds 5 and 6 showed weak cytotoxic activities against HCT116 cells with the mean IC50 values of 75.495 ± 4.29 and 44.326 ± 3.70 μM, respectively, in comparison with doxorubicin (positive control) with a mean IC50 value of 0.41 ± 0.02 μM. Compounds 1, 3, and 7 showed moderate cytotoxic activities against HepG2 cells with the mean IC50 values of 10.231 ± 1.04, 14.319 ± 2.08, and 12.731 ± 1.93 μM, respectively, while compounds 5, 6, 8, and 9 showed weak cytotoxic activities against HepG2 cells with the mean IC50 values of 79.824 ± 5.09, 67.632 ± 5.76, 98.637 ± 7.13, and 42.839 ± 3.33 μM, respectively, in comparison with doxorubicin with a mean IC50 value of 0.74 ± 0.06 μM [Table 2] and [Figure 2], [Figure 3].
|Table 2: The cytotoxic activity (half maximal inhibitory concentration) of 9 compounds against the growth of HCT116 and HepG2 cells|
Click here to view
|Figure 2:Morphological and cytological features of colorectal carcinoma cell line (HCT116) (a and b) treated with 1% dimethyl sulfoxide (vehicle control) (a), treated with compound 3 in a concentration of 100 μM (b) after 72 h of cell exposure to the investigated compound. (a) HCT116 grown as a control with dimethyl sulfoxide solvent, showing the characteristic monolayer of carcinoma cells, with the standard features of cellular atypia: nuclear and cytoplasmic pleomorphism, increased nucleus: cytoplasm ratio, highly irregularly-shaped cells (tadpole, caudate), irregular nuclear shapes, and hyperchromasia. (b) HCT116 with compound 3 addition shows a decreased number of colorectal carcinoma cells and necrotic cells in suspension|
Click here to view
|Figure 3:Morphological and cytological features of hepatocellular carcinoma cell line (HepG2) (a and b) treated with 1% dimethyl sulfoxide (vehicle control) (a), treated with compound 1 in a concentration of 100 μM (b) after 72 h of cell exposure to the investigated compound. (a) HepG2 grown as a control with dimethyl sulfoxide solvent, showing the characteristic monolayer of carcinoma cells, with the standard features of cellular atypia: nuclear and cytoplasmic pleomorphism, increased nucleus: cytoplasm ratio, highly irregularly-shaped cells (tadpole, caudate), irregular nuclear shapes, and hyperchromasia. (b) HepG2 with compound 1 addition shows a moderate decrease in the number of hepatocellular carcinoma cells and necrotic cells in suspension|
Click here to view
| Conclusion|| |
Nine natural compounds 1–9 were isolated from a medicinal plant, identified as X. strumarium . These compounds are derived from different metabolic pathways; interestingly, most of them showed an appreciated activity against HCT116 and HepG2 cancer cell lines.
The authors gratefully acknowledge the King Fahd Medical Research Center for giving them the opportunity to work in its central laboratory.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Lin R. Asteraceae. In: Flora of China; Science Press: Beijing 1979; pp. 324-325.
Qu J, Deng S, Li L, Liu Y, LiY, Ma S, Chen X, et al
. Cytotoxic dimeric xanthanolides from fruits of Xanthium chinense
. Phytochemistry 2016;132:115-22.
Bui VB, Liu ST, Zhu JJ, Xiong J, Zhao Y, Yang GX, et al
. Sesquiterpene lactones from the aerial parts of Xanthium sibiricum
and their cytotoxic effects on human cancer cell lines. Phytochem Lett 2012;5: 685-9.
Jiang H, Yang L, Ma GX, Xing XD, Yan ML, Zhang YY, et al
. New phenylpropanoid derivatives from the fruits of Xanthium sibiricum
and their anti-inflammatory activity. Fitoterapia 2017;117:11-5.
West PL, Mckeown NJ, Hendrickson RG. Muscle spasm associated with therapeutic use of Cang Er Zi Wan. Clin Toxicol 2010;48:380-4.
Han T, Li HL, Zhang QY, Han P, Zheng HC, Rahman K, et al
. Bioactivity-guided fractionation for anti-inflammatory and analgesic properties and constituents of Xanthium strumarium L
. Phytomedicine 2007;14:825-9.
Karmakar UK, Ishikawa N, Toume K, Arai MA, Sadhu SK, Ahmed F, et al
. Sesquiterpenes with TRAIL-resistance overcoming activity from Xanthium strumarium
. Bioorg Med Chem 2015;23:474654.
Xu F, Xiao C, Lv X, Lei M, Hu L. Two new Dimmeric xanthanolides
isolated from Xanthium mogolium
Kitag plant. Tetrahedron Lett 2017;58:1312-5.
Shang J, Hu B, Wang J, Zhu F, Kang Y, Li D, et al
. Cheminformatic Insight into the differences between terrestrial and marine originated natural products. J Chem Inf Model 2018;58:1182-93.
Shang J, Sun H, Liu H, Chen F, Tian S, Pan P, et al
. Comparative analyses of structural features and scaffold diversity for purchasable compound libraries. Cheminformatics 2017;9:1-16.
Ewing I, Hurley JJ, Josephides E, Millar A. The molecular genetics of colorectal cancer. Frontline Gastroenterol 2014;5:26-30.
Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, et al.
Cancer incidence and mortality worldwide: IARC Cancer Base No. 11. Lyon, France: International Agency for Research on Cancer 2013.
Abdul-Hameed ZH, Alarif WM, Omer AMS, El-Omri A, Ayyad S-EN, Badria FA, et al
. Selective Anti-proliferative activity of indole alkaloids from Rhazya stricta
decne leaves. Lett Org Chem 2019;16:941-7.
Ashwood VA, Buckingham RE, Cassidy F, Evans JM, Faruk EA, Hamilton TC, et al
. Synthesis and antihypertensive activity of 4-(cyclic amido)-2H-1-benzopyrans. J Med. Chem 1986;29:2194-201.
Bergmann R, Gericke R. Synthesis and antihypertensive activity of 4-(1,2-dihydro-2-oxo-1-pyridyl)-2H-1-benzopyrans and related compounds, new potassium channel activators. J Med Chem 1990;33:492-504.
Sam TW, Sutherland JK. Medium-ring 1,5-dienes. Part IV. Reactions of germacra-1(10),4,7(11)-triene with singlet oxygen and with the triphenyl phosphite-ozone adduct. J Chem Soc Perkin Trans 1975;1:2336-40.-
Choi JM, Lee KT, Ka H. Jung WT, Jung HJ, Park HJ. Constituents of the essential oil of the Cinnamomum cassia
stem bark and the biological properties. Arch Pharmacal Res 2001;24:418-23.
Choi JM, Lee EO, Lee HJ, Kim KH, Ahn KS, Shim BS, et al
. Identification of campesterol from Chrysanthemum coronarium L.
and its antiangiogenic activities. Phytother Res 2007;21:954-9.
WiIluhn G, Skibinski A, Schmidt TJ. Structure revision of Xanthalongin
and further Sesquiterpene lactones
from flowers of Arnica longifolia
. Planta Med 1998;64:635-9.
Bohlmann F, Jakupovic J, Schuster A. Further eudesmanolides and xanthanolides from Telekia speciose
. Phytochemistry 1981;20:1891-3.
Romero M, Zanuy M, Rosell E, Cascante M, Piulats J, Font-Bardia M, et al
. Optimization of xanthatin extraction from Xanthium spinosum L
. and its cytotoxic, anti-angiogenesis and antiviral properties. Eur J Med Chem 2015;90:491-6.
Bohlmann F, Zdero C. An isomer of xanthanol from Xanthium orientale
. Phytochemistry 1981;20:2429-30.
Alarif WM, Al-Saihati ZA, Ph MH, Abdel-Lateff A, Elfaky MA, Bawakid NO, et al
. Cytotoxic pyran-based Cembranoids from Sarcophyton glaucum.
Lett Org Chem 2018;15:967-71.
Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, et al
. Synthesis and antihypertensive activity of 4-(cyclic amido)-2H-1-benzopyrans. J Natl Cancer Inst 1990;82:1107-12.
Schmitz FJ, Bourden BF, Toth SI. Antitumor and cytotoxic compounds from marine organisms. In: Attaway DH, Zaborsky OR, editors. Marine Biotechnology, Pharmaceutical and Bioactive Natural Products. New York, NY, USA: Plenum Press: 1993. 1:198, 35.
Burger AM, Fiebig HH. Preclinical screening for new anticancer agents. In: Figg WD, McLeod HL, editors. Handbook of Anticancer Pharmacokinetics and Pharmacodynamics, Cancer Drug Discovery and Development. Totowa, NJ, USA: Humana Press Inc.; 2004. p. 36-7.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]