Cytotoxic Compounds from Aloe megalacantha

Phytochemical investigation of the ethyl acetate extract of the roots of Aloe megalacantha led to the isolation of four new natural products—1,8-dimethoxynepodinol (1), aloesaponarin III (2), 10-O-methylchrysalodin (3) and methyl-26-O-feruloyl-oxyhexacosanate (4)—along with ten known compounds. All purified metabolites were characterized by NMR, mass spectrometric analyses and comparison with literature data. The isolates were evaluated for their cytotoxic activity against a human cervix carcinoma cell line KB-3-1 and some of them exhibited good activity, with aloesaponarin II (IC50 = 0.98 µM) being the most active compound.


Introduction
Aloe is the largest genus in the Asphodelaceae family and comprised of more than 400 species [1], ranging from diminutive shrubs to large trees distributed across Africa, with the major diversity in South Africa [2]. Aloe is represented in East Africa by 83 species, of which 38 grow naturally in Ethiopia, including 15 endemic species [2][3][4]. It has long medicinal and economic history since 1500 BC [5]. The Aloe gel found in the interior of the leaves has been extensively used for the treatment of human and livestock diseases [6,7]. In fact, Aloe vera is well known as a commercial crop in many countries for its extensive use in cosmetic and pharmaceutical industries [6]. The plants produce different varieties of polyketides; mainly anthraquinones have been reported to possess antimicrobial [8], antimalarial [9], and cytotoxic [10] activities.
Aloe megalacantha is one of the species growing in Ethiopia and has been commonly used by traditional healers in the Western and Southern part of Ethiopia for the treatment of wounds. While the genus Aloe is the most extensively investigated species due to its rich medicinal value and chemotaxonomic aspect, no phytochemical analysis and biological activities of A. megalacantha have been reported. Thus, as part of our ongoing search for new bioactive natural products of plants from the Asphodelaceae family [11][12][13], we report the isolation of four new natural products 1-4, along with ten known compounds (one coumarin and nine anthraquinones). The cytotoxicity of the compounds against the human cervix carcinoma cell line KB-3-1 is also reported.
One of the methoxy groups (δ H 3.99, δ C 56.3) is present at C-8 (δ C 157.0) according to its long range HMBC correlation ( Figure 2) with the neighbouring carbons, C-7 (δ C 106.7) and C-8 (δ C 157.0). A broad singlet aromatic proton at δ H 7.48 (H-4) showed a long-range NOE interaction in the NOESY spectrum with H-5 (δ H 6.97) and the methyl group (δ H 2.30; δ C 19.0) located as expected according to biosynthetic considerations at C-3 (δ C 133.6). The substituents at C-1 (δ C 154.8) and C-2 (δ C 132.0) were, therefore, established to be the methoxy (δ H 3.73, δ C 64.1) and the hydroxymethylketone unit (δ H 4.55, δ C 70.5; δ C 207.5) respectively, based on a 3 J C,H HMBC correlation of both H-4 (δ H 7.48) and the hydroxymethyl protons (δ H 4.55) with C-2 (δ C 132.0). These placements were further confirmed by the long range HMBC correlations observed between methoxy protons (δ H 3.73) and its nearby C-1 and C-2 carbons. Interestingly, the down-field shifted signal at δ C 207.5 for the aryl ketone in the 13 C-NMR spectrum is consistent with a group being ortho-disubstituted, resulting in a deviation from planarity as a result of steric repulsion. Therefore, based on the above spectroscopic evidence, the new compound was characterized as 2-hydroxymethylacetyl-1,8-dimethoxy-3-methylnaphthalene, which was given the trivial name 1,8-dimethoxynepodinol (1). new compound was characterized as 2-hydroxymethylacetyl-1,8-dimethoxy-3-methylnaphthalene, which was given the trivial name 1,8-dimethoxynepodinol (1).  Compound 2 was isolated as yellow solid. The HR-ESI-MS provided a pseudomolecular ion peak at m/z 297.0753 [M + H] + , corresponding to the molecular formula of C17H12O5. The UV spectrum (λmax 264, 298, 336, 371 nm) and NMR spectra suggested an anthraquinone skeleton [23]. The 13 C-NMR spectrum in total contains seventeen carbon signals (Table 1), among them three carbonyl Compound 2 was isolated as yellow solid. The HR-ESI-MS provided a pseudomolecular ion peak at m/z 297.0753 [M + H] + , corresponding to the molecular formula of C 17 H 12 O 5 . The UV spectrum (λ max 264, 298, 336, 371 nm) and NMR spectra suggested an anthraquinone skeleton [23]. The 13 C-NMR spectrum in total contains seventeen carbon signals (Table 1), among them three carbonyl signals (δ C 184.0, 183.5 and 168.3). The former two signals were attributed to a quinone system and the latter to the carboxylic acid methyl ester. In the 1 H-NMR spectrum (Table 1), four mutually coupled aromatic protons of AA BB spin pattern centered at δ H 8.19 (dd, J = 7.7, 1.4 Hz, 1H), 7.86 (td, J = 7.5, 1.4 Hz, 1H), 7.91 (td, J = 7.5, 1.5 Hz, 1H) and 8.24 (dd, J = 7.7, 1.5 Hz, 1H) were assigned to H-5, H-6, H-7, and H-8, respectively, located at the disubstituted ring C. A singlet at δ H 7.75 was assigned to H-4 in ring A, which otherwise is fully substituted with a methyl group shifted downfield to δ H 2.71 due to the deshielding effect of the neighbouring carbonyl group attached to C-1 (δ C 142.3). Furthermore, a carboxylic acid methyl ester (δ H 3.95; δ C 52.9, δ C 168.3) at C-2 (δ C 130.8) (based on biosynthetic considerations) and a hydroxy group at C-3 (δ C 159.0). These assignments were in agreement with the biosynthetic consideration that appear to have been formed through folding of the octaketide chain in an unusual way as in aloesaponarin II [24] and further confirmed by the HMBC correlation ( . These data are consistent with the compound being methyl 3-hydroxy-1-methyl-9,10-dioxo-9,10-dihydroanthracene-2-carboxylate ( Figure 1) which was given the trivial name aloesaponarin III. This is the first report on the natural occurrence of compound 2 that had previously been reported as a synthetic intermediate [25].
The 1 H-NMR spectroscopic (Table 1) [14]. The HMBC spectrum ( Figure 3) showed a 3 J C,H correlation between H-6 (δ H 8.85) and C-10 (δ C 75.6) supporting the suggestion that C-7 is the point of attachment to the former half of the molecule. The only notable difference is the presence of a singlet integrating for three protons at δ H 2.87 attached to the carbon resonating at δ C 50.7 showing a HMBC correlation to C-10 (δ C 75.6). The upfield chemical shift value of the methoxy group (δ H 2.87) may be ascribed to anisotropy. It also showed an NOE ( Figure 3) correlation with H-4. The optical activity ([α] 20 D = +46 (c 0.5, CH 2 Cl 2 )) and the CD spectrum confirms the chirality of compound 3. The absolute configuration at the chiral center was then established in comparison with a related dimeric anthraquinone, abiyquinone B [26]. Compound 3 has opposite optical activity and a strong negative Cotton effect at shorter wavelength close to 235 nm in the CD spectrum. Based on the above spectroscopic evidence, the third compound is (S)-10-(chrysophanol-7 -yl)-10-methoxy-aloeemodin-9-anthrone that was given the trivial name  Hz. This indicated a trans configuration and the signals were assigned to H-2′ and H-3′, respectively. These protons showed HMBC correlations with the ester carbonyl carbon at δC 167.5, suggesting that these protons were part of an α,β-unsaturated ester moiety. The three aromatic proton signals at δH 7.03 (JHH = 1.9 Hz), 6.92 (JHH = 8.2 Hz) and 7.07 (JHH = 8.2, 1.9 Hz) were assigned to H-5′, H-8′, and H-9′, respectively based on their HH-COSY interactions and coupling constants. These assignments were further supported by the HMBC correlations of the downfield shifted proton H-3′ (δH 7.61) with C-5′ (δC 109.4) and C-9′ (δC 123.2) that in turn have HMQC correlations with H-5′ and H-9′, respectively. The 1 H-NMR spectrum further showed the presence of a singlet at δH 5.84 corresponding to a phenolic hydroxy group, two singlets integrating for three protons each at δH 3.66 and 3.93 for two methoxy groups, two triplets at δH 4.19 and 2.30 for methylene groups (attached to oxygen and carbonyl respectively, deduced from their chemical shift) and multiplets corresponding to long methylene chain. The 13 C-NMR spectrum contains signals corresponding to two ester carbonyl carbons (δC 174.5, 167.5), five aromatic/olefinic methine groups (δC 144.8, 123.2, 115.8, 114.8, 109.4), two oxygenated aromatic quaternary carbons (δC 148.0, 146.9), one oxymethylene (δC 64.8), two methoxy (δC 56.1, 51.6) and a number of methylene (34.3-24.9) carbons. The position of one methoxy group (δH 3.93; δC 56.1) was established at C-6′ (δC 148.0) according to its HMBC correlation to C-6′, while the hydroxy group at δH 5.84 showed a correlation to C-7′ (δC 146.9). The other methoxy group (δH 3.66; δC 51.6) corresponds to a methyl ester deduced from its upfield shifted (δC 51.6) signal [12] and from its HMBC correlation with the ester carbonyl (δC 174.5). Comparison of these spectroscopic data with the literature revealed that the compound was very similar to ω-feruloyloxyacid [27] and ethyl 24-(feruloyloxy)docosanoate [28]. The only difference was the absence of a methoxy group and the length of the aliphatic chain. The NMR spectra and ESI-MS (m/z 625 [M+Na] + , 601 [M−H] − ) were in accordance with the presence of a ω-oxygenated C26 fatty acid chain, like ω-feruloyloxyacids with along aliphatic chain (C19-C27), reported from peat soil [27]. Based on the spectroscopic data and the literature information, the structure of compound 4 was concluded to be methyl (E)-26-((3-(4hydroxy-3-methoxyphenyl)acryloyl)oxy)hexacosanoate that was given the trivial name methyl 26-Oferuloyl-oxyhexacosanoate.
The isolates were evaluated for their cytotoxic activities against the human cervix cancer cell line KB-3-1, with cryptophycin-52 (IC50 = 1.3 × 10 −5 µM) and griseofulvin (IC50 = 19.0 µM) as positive controls, as described in previous reports [29]. Two compounds, aloesaponarin II (11) and aloesaponarin I (12) showed good cytotoxic activity with IC50 values of 0.98 µM and 16.00 µM, respectively whereas the dimeric anthraquinone asphodelin (14) showed weak activity (>60.00 µM). The other compounds showed little or no inhibitory activities. The activity of aloesaponarin II is sixteen times higher compared to aloesaponarin I; however, both possess similar quinone nuclei with a methyl and hydroxyl substitutions at similar position. The only difference is the presence of an electron withdrawing  Hz. This indicated a trans configuration and the signals were assigned to H-2 and H-3 , respectively. These protons showed HMBC correlations with the ester carbonyl carbon at δ C 167.5, suggesting that these protons were part of an α,β-unsaturated ester moiety. The three aromatic proton signals at δ H 7.03 (J HH = 1.9 Hz), 6.92 (J HH = 8.2 Hz) and 7.07 (J HH = 8.2, 1.9 Hz) were assigned to H-5 , H-8 , and H-9 , respectively based on their HH-COSY interactions and coupling constants. These assignments were further supported by the HMBC correlations of the downfield shifted proton H-3 (δ H 7.61) with C-5 (δ C 109.4) and C-9 (δ C 123.2) that in turn have HMQC correlations with H-5 and H-9 , respectively. The 1 H-NMR spectrum further showed the presence of a singlet at δ H 5.84 corresponding to a phenolic hydroxy group, two singlets integrating for three protons each at δ H 3.66 and 3.93 for two methoxy groups, two triplets at δ H 4.19 and 2.30 for methylene groups (attached to oxygen and carbonyl respectively, deduced from their chemical shift) and multiplets corresponding to long methylene chain. The 13 C-NMR spectrum contains signals corresponding to two ester carbonyl carbons (δ C 174.5, 167.5), five aromatic/olefinic methine groups (δ C 144. 8, 123.2, 115.8, 114.8, 109.4), two oxygenated aromatic quaternary carbons (δ C 148.0, 146.9), one oxymethylene (δ C 64.8), two methoxy (δ C 56.1, 51.6) and a number of methylene (34.3-24.9) carbons. The position of one methoxy group (δ H 3.93; δ C 56.1) was established at C-6 (δ C 148.0) according to its HMBC correlation to C-6 , while the hydroxy group at δ H 5.84 showed a correlation to C-7 (δ C 146.9). The other methoxy group (δ H 3.66; δ C 51.6) corresponds to a methyl ester deduced from its upfield shifted (δ C 51.6) signal [12] and from its HMBC correlation with the ester carbonyl (δ C 174.5). Comparison of these spectroscopic data with the literature revealed that the compound was very similar to ω-feruloyloxyacid [27] and ethyl 24-(feruloyloxy)docosanoate [28]. The only difference was the absence of a methoxy group and the length of the aliphatic chain. The NMR spectra and ESI-MS (m/z 625 [M + Na] + , 601 [M−H] − ) were in accordance with the presence of a ω-oxygenated C 26 fatty acid chain, like ω-feruloyloxyacids with along aliphatic chain (C 19 -C 27 ), reported from peat soil [27]. Based on the spectroscopic data and the literature information, the structure of compound 4 was concluded to be methyl (E)-26-((3-(4-hydroxy-3-methoxyphenyl)acryloyl)oxy)hexacosanoate that was given the trivial name methyl 26-O-feruloyl-oxyhexacosanoate.
The isolates were evaluated for their cytotoxic activities against the human cervix cancer cell line KB-3-1, with cryptophycin-52 (IC 50 = 1.3 × 10 −5 µM) and griseofulvin (IC 50 = 19.0 µM) as positive controls, as described in previous reports [29]. Two compounds, aloesaponarin II (11) and aloesaponarin I (12) showed good cytotoxic activity with IC 50 values of 0.98 µM and 16.00 µM, respectively whereas the dimeric anthraquinone asphodelin (14) showed weak activity (>60.00 µM). The other compounds showed little or no inhibitory activities. The activity of aloesaponarin II is sixteen times higher compared to aloesaponarin I; however, both possess similar quinone nuclei with a methyl and hydroxyl substitutions at similar position. The only difference is the presence of an electron withdrawing methyl ester group in aloesaponarin I, which may negatively influence the cytotoxic activity of the compound.
It is worth mentioning that several cancer chemotherapeutic agents such as doxorubicin, mitomycin C, and mitoxantrone contain a common structural quinone nucleus. This chemical structure allows them to be involved in multiple biological oxidative processes [30]. The reduction of the quinone leads to toxic species (semiquinone anion radical and hydroquinone) which act selectively in hypoxic tissues like tumor cells [31].

Plant Materials
The roots of A. megalacantha were collected from West Arsinegele (7 • 22 26.2" N 38 • 40 02.6" E), Ethiopia, 240 Km away from Addis Ababa on the way to Shashemene in September 2016. The plant material was identified by professional botanist at Jimma University (Dr. Kitessa Hundera) and the voucher specimen (voucher number NA-07/16) has been deposited in the Jimma University Herbarium.

Extraction and Isolation
The air dried and powdered roots of A. megalacantha (840 g) were extracted exhaustively with ethyl acetate (3 L) four times each for 24 h at room temperature. The extract was then concentrated under reduced pressure using a rotary evaporator to yield 34 g of brown extract. A portion of the extract (30 g) was subjected to column chromatography on oxalic acid deactivated silica gel (400 g) and the column was eluted with petroleum ether containing increasing amounts of ethyl acetate to give 37 fractions each of ca. 250 mL.

Cytotoxicity Assay
The human cervix carcinoma cell line KB-3-1 was used in the cytotoxicity assay as previously described [29]. The cell line was cultivated as a monolayer in DMEM (Dulbecco's modified Eagle medium) with glucose (4.5 g/L), L-glutamine, sodium pyruvate and phenol red, supplemented with 10% fetal bovine serum (FBS) and were maintained at 5.3% CO 2 and 37 • C in humidified air. The cells at 70% confluence were detached with trypsin-ethylenediamine tetraacetic acid solution (0.05%; 0.02% in DPBS) and placed in sterile 96-well plates in a density of 10,000 cells in 100 µL medium per well. The dilution series of the compounds was prepared from stock solutions in DMSO of concentrations of 100 mM, 50 mM or 25 mM and the stock solutions were diluted with culture medium (10% FBS) down to pM concentrations. The dilution prepared from stock solution was added to the wells and each concentration was tested in at least six replicates. The control contained the same concentration of DMSO as the first dilution. After incubation for 72 h at 37 • C and 5.3% CO 2 -humidified air, 30 µL of an aqueous resazurin solution (175 µM) was added to each well. The cells were incubated at the same conditions for 5 h. Subsequently, the fluorescence was recorded at a wavelength of 588 nm. The IC 50 values were calculated as a sigmoidal dose response curve using Graphpad Prism 4.03 (Graphpad software Inc., San Diego, CA, USA).

Conclusions
Four new natural products along with ten other known compounds were identified from the root of A. megalacantha. The two aloesaponarins (aloesaponarin I and II) showed good cytotoxic activity even greater than one of the reference drug (griseofulvin) against KB-3-1 human cervical cancer cell line, with the highest activity observed for aloesaponarin II.