Two new cytotoxic furoquinoline alkaloids isolated ...

Natural Product Research Formerly Natural Product Letters

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Two new cytotoxic furoquinoline alkaloids isolated from Aegle marmelos (Linn.) Correa Magdy M. D. Mohammed, Nabaweya A. Ibrahim, Fatma S. El-Sakhawy, Khaled M. Mohamed & Doaa A.-H. Deabes To cite this article: Magdy M. D. Mohammed, Nabaweya A. Ibrahim, Fatma S. El-Sakhawy, Khaled M. Mohamed & Doaa A.-H. Deabes (2016) Two new cytotoxic furoquinoline alkaloids isolated from Aegle marmelos (Linn.) Correa, Natural Product Research, 30:22, 2559-2566, DOI: 10.1080/14786419.2015.1126262 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1126262

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Date: 04 November 2016, At: 00:53

Natural Product Research, 2016 VOL. 30, NO. 22, 2559–2566 http://dx.doi.org/10.1080/14786419.2015.1126262

Two new cytotoxic furoquinoline alkaloids isolated from Aegle marmelos (Linn.) Correa Magdy M. D. Mohammeda,b , Nabaweya A. Ibrahimb, Fatma S. El-Sakhawyc, Khaled M. Mohamedb and Doaa A.-H. Deabesb a

Nucleic Acid Center, Institute of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense M, Denmark; bPharmaceutical and Drug Industries Research Division, Pharmacognosy Department, National Research Centre, Cairo, Egypt; cFaculty of Pharmacy, Pharmacognosy Department, Cairo University, Giza, Egypt

ABSTRACT

Two new cytotoxic furoquinoline alkaloids were isolated from the leaves of Aegle marmelos (Linn.) Correa; one from the total alkaloidal fraction (acid/base shake-out method) of the CHCl3 extract and identified as 7,8-dihydroxy-4-hydrofuroquinoline and named trivially as Aegelbine-A. The other new alkaloid isolated from the pet. ether extract and identified as 4-hydro-7-hydroxy-8-prenyloxyfuroquinoline and named trivially as Aegelbine-B, together with a known alkaloid; aegeline and a known phenolic acid; ρ-hydroxybenzoic acid. The structures of all the isolated compounds were established based on 1D and 2D NMR spectroscopy and HR-ESI/MS. The cytotoxic activity of the isolated compounds was evaluated in vitro against HepG-2, PC3, A549 and MCF-7 cell lines. The obtained results revealed promising activity with structure-based relationship which is discussed briefly. HH H

3

4a 7

3a

8a

8

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Aegelbine-A

HH

H 4

5 6

8

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C

5´

3´

CH 4´

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N 9

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O

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O

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4a 7

H O

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KEYWORDS

Aegle marmelos (Linn.) Correa; rutaceae; furoquinoline alkaloids; cytotoxicity; structure– activity relationship studies

H 4

5 6

H O

ARTICLE HISTORY

Received 10 August 2015 Accepted 2 November 2015

2´

Aegelbine-B

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1. Introduction Aegle marmelos (Linn.) Correa belongs to the family Rutaceae, it has different common names i.e. Bael, Golden Apple, Oranger du Malabar, etc. which is varied according to its origin (Orwa et al. 2009). It is distributed wildly throughout tropical Asia (sub-Himalayan tract, central and Southern India and can be cultivated in Northern region) and Africa (Jagetia et al. 2005; Kala 2006; Kesari et al. 2006). A. marmelos considered as one of the most important medicinal CONTACT Magdy M. D. Mohammed [email protected] Supplemental data for this article can be accessed at http://dx.doi.org/10.1080/14786419.2015.1126262. © 2015 Informa UK Limited, trading as Taylor & Francis Group

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plants in India, Burma and Ceylon. It has a long history of medicinal use in the Ayurvedic system for the treatment of various ailments, i.e. intermittent fevers, fertility control, treatment after childbirth and fish poison (Basu & Sen 1974). Earlier reports of the phytoconstituents of A. marmelos leaves, fruits and root bark revealed the presence of coumarins (Basu & Sen 1974; Chatterjee et al. 1978; Ali & Pervez 2004), alkaloids i.e. furoquinoline (skimmianine) (Shoeb et al. 1973; Basu & Sen 1974; Manandhar et al. 1978; Sharma et al. 1981; Govindachari & Premila 1983; Nouga et al. 2016), sterols and essential oils (Dhankhar et al. 2011; Ibrahim et al. 2015). Furthermore, newly studies proved that the plant has wide spectrum of biological activities i.e. insecticidal, antidiabetic, anti-inflammatory, antifungal and anticancer (Samarasekera et al. 2004; Maity et al. 2009; Ibrahim et al. 2015). The aims of the presented study were to review the phytochemical constituents of A. marmelos searching for the possible presence of new alkaloids. Which accompanied by an evaluation of the cytotoxic activity of the isolates, and then to highlight their potentials as candidates for new drugs that may be of value in the treatment and prevention of human and livestock diseases. Extensive chromatographic separation and purification successfully resulted in the isolation of two new alkaloids of the furoquinoline type, identified as 7,8-dihydroxy-4-hydrofuroquinoline and 4-hydro-7-hydroxy-8-prenyloxyfuroquinoline, they were commonly named as aegelbine-A and aegelbine-B, respectively, along with a known alkaloid aegeline and a phenolic acid ρ-hydroxybenzoic acid. The cytotoxic activity of all isolates was carried out against HepG-2, MCF-7, PC3 and A549, using MTT assay in comparison with Adriamycin (Doxorubicin). The obtained results revealed strong to moderate activity with some selectivity of the isolated compounds against the tested cell lines.

2. Results and discussion The preliminary phytochemical screening of both leaves and fruits of A. marmelos, suggested the presence of alkaloids with a total content of 1.4 and 1.3% (w/w), respectively. Chromatographic analysis of the leaves (pet. ether and CHCl3) extracts for the possible presence of alkaloids suggested further phytochemical fractionation, separation and purification (see Experimental part). Further extensive chromatography resulted in the isolation of two new furoquinoline alkaloids; one from the total alkaloidal fraction (acid/base shake-out method) of CHCl3 extract, which is identified as 7,8-dihydroxy-4-hydrofuroquinoline and named trivially as Aegelbine-A, while the other new alkaloid isolated from the pet. ether extract and identified as 4-hydro-7-hydroxy-8-prenyloxyfuroquinoline and named trivially as Aegelbine-B, together with a known alkaloid; aegeline (Riyanto et al. 2001), and a known phenolic acid; ρ-hydroxybenzoic acid (Dhakal et al. 2009). All the isolates were structurally elucidated using 1D-, 2D-NMR, HR-ESI/MS and by comparison with the literature.

2.1. Compound (1) It was isolated from the total alkaloidal fraction prepared from the CHCl3 extract as orange red crystals, with m.p. 230–232 °C (MeOH). Its molecular formula was determined to be C11H7NO3 using HRESI/MS (+ve, S7), which revealed the presence of quasi-molecular ion peak at m/z 225.0396 [M + H + Na]+ (Calcd 225.0402 for C11H8NO3Na). 1H- and 13C-NMR (CD3OD, S1 and S2, respectively) spectra suggested a furoquinoline alkaloid base skeleton (Ayafor & Okogun

Natural Product Research

H H

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CH3 4´

Figure 1. The established structures of the new isolated alkaloids (1 and 2).

1982). Its 13C-NMR (S2) showed the presence of 11 signals, resolved by DEPT (S4) and HSQC (S5) as five methines and six quaternary carbons. The 1H-NMR spectrum displayed a pair of AB doublets appeared at δH 6.85 and δH 7.76 (each 1H, d, J = 2.8 Hz; H-3 and H-2, respectively) corresponding to the two furan protons, which correlated with a cross-peak as appeared in COSY spectrum (S3), moreover, another cross-peak assigned to the two ortho-coupled aromatic protons, that appeared at δH 6.33 and δH 7.94 (each 1H, d, J = 9.6 Hz) and were assigned to H-6 and H-5, respectively. Furthermore, a sharp singlet signal appeared at δH 7.29 (1H, s) corresponding to H-4 of the quinoline ring, confirming the absence of the characteristic methoxy group at C-4 for the furoquinoline base skeleton (Ayafor & Okogun 1982). 1H-13C HMBC experiment (S6) confirmed the proposed structure of compound (1) by clarifying a set of cross-peaks revealed the long-range correlations; the two furan ring protons H-2 with C-3 and C-3a and H-3 with C-2, C-4 and C-3a, while H-4 correlated with C-3, C-3a, C-5, C-4a and C-8a, H-5 with C-4, C-4a, C-6, C-7 and C-8a, and H-6 with C-4a, C-5 and C-7. These findings unambiguously established the structure of compound (1) as depicted in Figure 1 and assigned to 7,8-dihydroxy-4-hydrofuroquinoline and trivially named as aegelbine-A.

2.2. Compound (2) It was isolated from the pet. ether extract of the leaves as yellowish white needles, with m.p. 86–88 °C (CHCl3). Its molecular formula was determined to be C16H15NO3 using HRESI/ MS (+ve, S14), which showed two quasi-molecular ion peaks at m/z 271.1209 [M + 2H]+ and m/z 293.1027 [M + H + Na]+ (Calcd 271.1208 for C16H17NO3 and 293.1022 for C16H16NO3Na). 1 H- and 13C-NMR spectra (CDCl3, S8 and S9, respectively) suggested a furoquinoline alkaloid base skeleton (Ayafor & Okogun 1982). Its 13C-NMR spectrum showed the presence of 16 signals, resolved by DEPT (S11) and HSQC (S12) as two methyls, one methylene, six methines and seven quaternary carbons. 1H-NMR (S8) and 1H-1H COSY spectra (S10) displayed a pair of AB doublets characteristic for the two furan protons, correlated together with a strong cross-peak and appeared at δH 6.82 and δH 7.69 (each 1H, d, J = 2.8 Hz) corresponding to H-3 and H-2, respectively. Moreover, another cross-peak assigned to the two ortho-coupled aromatic protons at δH 6.37 and δH 7.77 (each 1H, d, J = 9.6 Hz) corresponding to H-6 and H-5,

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respectively. Furthermore, a sharp singlet signal appeared at δH 7.36 (1H, s) corresponding to H-4 of the quinoline ring, confirming the absence of the characteristic methoxy group at C-4 for the furoquinoline base skeleton (Ayafor & Okogun 1982). In addition to that, a cross-peak represented the correlation of the olefinic proton appeared at δH 5.61 (1H, t, J = 7.6 Hz, H-2′) with the two oxymethylene protons appeared at δ 5.00 (2H, d, J = 7.6 Hz, H-1′), two additional cross-peaks were also observed and revealed the allylic correlations of the two vinylic methyl groups appeared at δH 1.72 and δH 1.74 (each 3H, brs) with the olefinic proton at δH 5.61 and with the two oxymethylene protons appeared at δH 5.00, assigned to prenyloxy side chain. The 1H-13C HMBC experiment (S13) confirmed the proposed structure of compound (2) by clarifying the long-range correlations (2J and 3J) as follow; a set of cross-peaks observed in the HMBC spectrum revealed long-range correlations of the two furan protons H-2 with C-3 and C-3a, and H-3 with C-2, C-3a and C-4. Furthermore, H-4 correlated with C-3, C-3a, C-4a, C-5 and C-8a, H-5 with C-4, C-4a, C-6, C-7 and C-8a and H-6 with C-4a, C-5 and C-7. Moreover, the attachment position of the prenyloxy group was confirmed to be at C-8 based on the HMBC long-rang correlation of the oxymethlene protons CH2-1′at δH 5.00 with C-8 at δC 148.57. These findings collectively established the structure of compound (2) as formulated in Figure 1 and assigned to 4-hydro-7-hydroxy-8-prenyloxyfuroquinoline and trivially named as aegelbine-B. The cytotoxic effects of all isolates were tested in vitro against HepG-2, PC-3, A549 and MCF-7 cell lines. The cytotoxicity data are shown (S15), and the clinically applied anticancer agent Adriamycin was used as a reference compound. The obtained results revealed that all compounds possessed cytotoxic activity against the tested cell lines, with different percentages of cell inhibition at 100 ppm, which reflects some sort of selectivity. In brief, all the tested compounds inhibited A549 cells with 15.3–36.5% when tested at 100 ppm, while ρ-hydroxybenzoic acid recorded a slight cell inhibition with 32.2, 32.7 and 50.7% at 100 ppm for A549, HepG-2 and MCF-7 cells, respectively, and it was completely inactive against PC-3 cells. Furthermore, aegeline and aegelbine-B were of some selectivity as they showed the highest inhibition (72.5 and 78.5%) against MCF-7 with IC50 values of 73.7 and 63.5 μg/mL, respectively, in addition to some inhibition that was recorded against the other cells (S15). Among the tested samples, both of the total alkaloidal fraction and aegelbine-A were the most interested ones, and exhibited the highest inhibition % against the different cells, briefly; the total alkaloidal fraction was cytotoxic to HepG-2 and MCF-7 with IC50 values of 52.9 and 59.5 μg/mL, respectively. Moreover, aegelbine-A was the most active against the tested cells HepG-2, PC-3 and MCF-7 with IC50 values of 71.4, 72.5 and 56.5 μg/mL, respectively. Many alkaloids are infamous because of their strong toxicity towards animals and humans. Most of the deadly alkaloids fall into the class of neurotoxins. The others have cytotoxic properties. A cytotoxic effect can be generated when cell membranes are made leaky (i.e. by saponins or steroidal alkaloids), or when elements of the cytoskeleton are inhibited (Chen et al. 2003). Disturbances of the cytoskeleton, DNA replication and DNA topoisomerase, or DNA alkylation and intercalation usually lead to cell death by apoptosis (Wink 1993). Planar and lipophilic alkaloids i.e. furoquinoline alkaloids are intercalating compounds that interact reversibly with the DNA double helix. Thus, the activity of the isolated alkaloids (i.e. furoquinoline type) in the presented study may be attributed to their ability to assemble between the stacks of paired nucleotides in the DNA double helix, and form covalent adducts


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with DNA bases (the so-called alkylating agents), that lead to apoptosis (Wink 1993; Roberts & Wink 1998; Wink et al. 1998; Wink 2007). The structural features of intercalating agents are planar polyaromatic systems which bind by insertion between DNA base-pairs, with a marked preference for 5-pyrimidine-purine-3 steps. The chromophores are linked to basic chains that might also play an important role in the affinity and selectivity shown by these compounds (Brana et al. 2001). There are two interactions that takes place: in the major and minor grooves. Interactions in the minor groove are composed of hydrogen bonding and hydrophobic effects. Interactions in the major groove are composed of hydrogen bonding and hydrophobic interactions as well as water-mediated interactions (Rohs et al. 2010). Due to the fact that hydrogen bonds in the major groove allow greater specificity due to the presence of four base pairs and the unique pattern between hydrogen bond donors and hydrogen bond acceptors (Rohs et al. 2010). It supports the obtained results that aegelbine-A with hydrogen donor (phenolic di-ortho-OH groups) and a planar aromatic system exhibited better potency and selectivity against MCF-7, HepG-2 and PC-3 more than aegelbine-B, which have only one phenolic OH group, besides the presence of the prenyloxy group in aegelbine-B which may cause some steric hindrance for its ability to bind covalently with DNA bases (Mohammed et al. 2012). Furthermore, the activity of aegeline against MCF-7 with IC50 value of 73.7 μg/mL may be attributed to its potential Michael acceptor activity that can lead to cell damage and cytotoxicity due to the presence of α,β-unsaturated carbonyl group (Amslinger 2010).

3. Experimental 3.1. Plant material The fresh plant Aegle marmelos (L.) Correa (leaves and fruits) was collected from in El-Zohrya botanical garden, Giza, Egypt. Fresh leaves were collected in April 2008 during flowering stage and fresh mature fruits were collected in February 2008. The plant was identified by Mrs Threase Labib consultant of plant taxonomy at the Ministry of Agriculture. A voucher specimen (No. 000171Ac 04-02-05-17) was kept at the Herbarium of El-Orman Botanical Garden. The plant materials were dried at room temperature and grounded into fine powder for further processing.

3.2. General Melting points (uncorrected) were determined on a Koffler’s melting point apparatus. NMR: Spectra were obtained using a pulse sequence supplied from Bruker AVANCE-III-400 MHz NMR spectrometer for (1D- and 2D-NMR). Chemical shifts were given in values (ppm) relative to trimethylsilane (TMS) as an internal reference for both carbon and proton. HR-ESI/MS: as reported (Mohammed et al. 2014). All solvents used were of AR grade. Kiesel gel 60 F254 (Merck) was used for analytical TLC.

3.3. Extraction and isolation 3.3.1 Quantitative estimation of the total alkaloidal content of both leaves and fruits of A. marmelos (L.) Correa The total alkaloidal content was estimated according to Harborne (1973), and resulted with 1.4 and 1.3% (w/w) of leaves and fruits, respectively.

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3.3.2. Extraction and isolation of alkaloids from the pet. ether extract of both leaves and fruits The powdered leaves and fruits (1 kg, each) were extracted with pet. ether using Soxhlet apparatus to afford (32.5 and 25 g), respectively. Chromatographic comparison of the pet. ether extracts of both leaves and fruits was performed on TLC using different solvent systems [CHCl3–MeOH (98:2 v/v, S1); C6H6–EtOAc–HCOOH (5:4:1v/v, S2); CHCl3– MeOH–H2O (65:45:5 v/v, S3)] and visualised by Dragendorff’s reagent. The obtained results confirmed the same TLC profile with the same/nearly the same spots for both leaves and fruits. Leaves extract was chosen for chromatography using preparative TLC (S1–S3) which revealed the presence of three distinct bands, two of which gave positive Dragendorff’s reagent. Further chromatographic separation, isolation and purification of the pet. ether of the leaves (10 g) was achieved by subsequent prep. TLC (S1–S3) led to the isolation of Aegeline as white crystals (25 mg), with m.p. 173–175 °C, it is freely soluble in CHCl3, pet. ether and insoluble in alcohol, its Rf values were 0.7, 0.63 and 0.76 using solvent systems (S1–S3), respectively. Aegelbine-B was isolated as yellowish white needles (20 mg) with m.p. 83–88 °C, it is freely soluble in CHCl3, ether and insoluble in alcohol, its Rf values were 0.8, 0.52 and 0.82 using solvent systems (S1–S3), respectively. Finally ρ-hydroxybenzoic acid was isolated as white crystal needles (15 mg), with m.p. 213–215 °C, it is slightly soluble in H2O, CHCl3 and soluble in alcohol, its Rf values were 0.6, 0.56 and 0.67 using solvent systems (S1–S3) respectively. All the isolated compounds were further purified by subsequent prep. TLC. 3.3.3. Extraction of crude alkaloids of Aegle marmelos (L.) Correa leaves The powdered leaves of A. marmelos (400 g, each) were grounsded separately, defatted with pet. ether (60–80 °C) several times, then the remaining powder was extracted with chloroform. The combined CHCl3 extracts were evaporated to dryness, and then the residue (10 g) was subjected to ‘acid–base checkout’ method (Mohammed et al. 2012) to afford (1 g) of the N-containing alkaloids ‘alkaloidal fraction’, which upon extensive separation and purification using prep. TLC with solvent systems (S1–S3) revealed the presence of only one major spot with Rf values of 0.48, 0.34 and 0.51, respectively, which was isolated and purified to afford (15 mg) of orange red crystals (aegelbine-A). 3.3.4. 7,8-dihydroxy-4-hydrofuroquinoline (1) Orange red crystals (15 mg), m.p. 230–232 °C (MeOH). 1H-NMR (CD3OD, 400 MHz) δH 6.85 (1H, d, J = 2.8 Hz, H-3), δH 7.76 (1H, d, J = 2.8 Hz, H-2), δH 6.33 (1H, d, J = 9.6 Hz, H-6), δH 7.94 (1H, d, J = 9.6 Hz, H-5), δH 7.29 (1H, s, H-4). 13C-NMR δC 147.94 (C-2, CH), δC 107.73 (C-3, CH), δC 127.08 (C-3a, C), δC 111.07 (C-4, CH), δC 117.47 (C-4a, C), δC 147.26 (C-5, CH), δC 114.73 (C-6, CH), δC 140.72 (C-7, C), δC 146.87 (C-8, C), δC 131.44 (C-8a, C), δC 162.99 (C-9, C). 3.3.5. 4-hydro-7-hydroxy-8-prenyloxyfuroquinoline (2) Yellowish white needles (20 mg), m.p. 86–88 °C (CHCl3). 1H-NMR (CDCl3, 400 MHz) δH 6.82 (1H, d, J = 2.8 Hz; H-3), δH 7.69 (1H, d, J = 2.8 Hz, H-2), δH 6.37 (1H, d, J = 9.6 Hz, H-6) and δH 7.77 (1H, d, J = 9.6 Hz, H-5), δH 7.36 (1H, s, H-4), δH 5.61 (1H, brt, J = 7.6 Hz, H-2′), δ 5.00 (2H, d, J = 7.6 Hz, H-1′), δH 1.72 and δH 1.74 (each 3H, brs). 13C-NMR δC 146.58 (C-2, CH), δC 106.68 (C-3, CH), δC 125.83 (C-3a, C), δC 113.13 (C-4, CH), δC 116.45 (C-4a, C), δC 144.232 (C-5, CH), δC 114.65 (C-6, CH), δC 143.78 (C-7, C), δC 148.57 (C-8, C), δC 131.63 (C-8a, C), δC 160.49 (C-9, C), δC 70.13 (C-1′, CH2), δC 119.74 (C-2′, CH), δC 139.71 (C-3′, C), δC 25.79 (C-4′, CH3), δC 18.09 (C-5′, CH3).


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3.4. Cytotoxic assay Detailed method as reported (Thabrew et al. 1997; Mohammed et al. 2013).

4. Conclusion The observed activities for the isolated new alkaloids corroborated those previously reported on similar furoquinoline base skeleton, indicating that the isolated compounds can be chemically explored to develop other chemotherapeutic agents.

Supplementary material Supplementary material relating to this article is available online, including original NMR and HR-ESI/ MS data (S1–S14), alongside the cytotoxicity data (S15).

Acknowledgement The authors are grateful to University of Southern Denmark, Odense, Denmark, for providing the facilities for instrumental analysis.

Disclosure statement No potential conflict of interest was reported by the authors.

ORCID Magdy M. D. Mohammed

http://orcid.org/0000-0001-5606-3498

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