Cytotoxic new furoquinoline alkaloid isolated from Ammi majus L. growing in Egypt

Abstract Two alkaloids of the furoquinoline-type were isolated from Ammi majus L.; a new one and was identified as 4-hydro-7-hydroxy-8-methoxyfuroquinoline (1), and the other was isolated for the second time from nature and was identified as 4-hydro-7-hydroxy-8-prenyloxyfuroquinoline (2). The structures of the isolated compounds were established and confirmed by 1D and 2D NMR spectroscopy including 1H, 13C NMR, COSY, HSQC and HMBC, while the exact masses were confirmed by HRESI/MS. The cytotoxic activity of the isolated compounds (1 and 2) was evaluated against HepG-2, PC-3, A-549 and MCF-7 and the obtained results suggested selective antiproliferative and cytotoxic effects, with IC50 = 230.2 and 326.5 μM against HepG-2 and MCF-7, respectively, for compound (1). While, compound (2) recorded IC50 = 234.2 μM against MCF-7.


Introduction
Ammi majus L. (Bishop's weed) closely resembles Ammi visnaga L. It belongs to the family Umbelliferae (Apiaceae). A. majus is an annual herbaceous plant which is widely spread in Egypt alongside the Nile and delta region (Fahmy et al. 1947). It is distributed in the Mediterranean region of Europe, western Asia and now cultivated in India (CCRUM 1987). A. majus is considered as one of the most important medicinal plants in Egypt (Singab 1998 as an emmenagogue to regulate menstruation, as a diuretic, and for the treatment of leprosy, kidney stones and urinary tract infections (NAPRALERT, 2014). Its fruits are taken internally by the public to cure leukoderma (Fahmy et al. 1947).
To date there are no reports of alkaloidal contents of A. majus although it has been reported as one of the plants containing alkaloids of the family Umbelliferae (Raffauf 1996), and was previously reported by Bick (1996) to give a positive alkaloidal test. Moreover, Hartwell (1982) reported that Ammi species, especially A. majus used in the folkloric medicine for the treatment of different types of tumours, i.e. spleen, uvula and fauces and in the induration of liver and stomach. This what prompted us to investigate its alkaloidal content.
Extensive chromatography of the MeOH extract of A. majus successfully resulted in the isolation of two alkaloids of the furoquinoline-type; where compound (1) the hitherto unknown was identified as 4-hydro-7-hydroxy-8-methoxyfuroquinoline, while, compound (2), was isolated for the second time from nature (firstly reported by Mohammed et al. 2016) and identified as 4-hydro-7-hydroxy-8-prenyloxyfuroquinoline. The isolated alkaloids were structurally elucidated by extensive NMR spectroscopy, and then were investigated for their cytotoxicity against HepG-2, PC-3, A-549 and MCF-7 cell lines. Structure activity relationship of the biologically active metabolites was studied in order to highlight their potentials as cytotoxic candidates for new drugs that may be helpful for human and livestock diseases.

Results and discussion
The defatted MeOH extract of A. majus was subjected to successive silica gel column chromatography to afford two alkaloids of the furoquinoline-type (1 and 2).

Compound (1)
It was isolated from fraction (V) by 100% CH 2 Cl 2 , as an amorphous solid, with molecular formula determined to be C 12 H 9 NO 3 using HRESI/MS (+ve, S7), which revealed the presence of two quasi-molecular ion peaks at m/z 217.0733 [M + 2H] + and m/z 239.0553 [M + H + Na] + (calcd 217.0738 for C 12 H 11 NO 3 and 239.0558 for C 12 H 10 NO 3 Na). 1 H and 13 C NMR (CDCl 3 , S1 and S2, respectively) spectra suggested a furoquinoline-type alkaloid base skeleton (Ayafor & Okogun 1982;Mohammed et al. 2016). Its 13 C NMR (S2) spectrum revealed the presence of 12 signals, classified by DEPT (S4) and HSQC (S5) spectra into one methyl, five methines and six quaternary carbons, which confirmed the molecular formula C 12 H 9 NO 3 . 1 H NMR spectrum revealed the presence of a downfield sharp singlet signal at δ H 4.27 (3H, s, -OCH 3 ) characteristic for a phenolic methoxy group. In addition, a pair of AB doublets appeared at δ H 6.80 and δ H 7.67 (each 1H, d, J = 2.0 Hz; H-3 and H-2, respectively) which are characteristic for the two furan ring protons, and confirmed via a cross-peak's correlation as appeared in COSY spectrum (S3), moreover, another cross-peak was assigned to the two ortho-coupled aromatic protons, that appeared at δ H 6.34 and δ H 7.75 (each 1H, d, J = 9.6 Hz; H-6 and H-5, respectively). Furthermore, the sharp singlet signal that appeared at δ H 7.33 (1H, s) was assigned to H-4 based on the HMBC correlation of H-4 with C-3 at δ C 106.66 and with C-5 at δ C 144.34 ( Figure S17).
The postulated structure of compound (1) was confirmed through HMBC correlations (S6) as appeared clearly in Figure S17; briefly, H-3 with C-3a and C-4, H-4 with C-3, C-3a and C-4a. H-5 and H-6 were distinguished on the base that H-5 showing HMBC correlation to C-4 at δ C 112.86, in addition, the HMBC correlation of H-5 to δ C 139.40 allowed this resonance to be assigned to C-7, which declared that C-8 resonance to be assigned at δ C 145.04. Moreover, the downfield phenolic methoxy group was confirmed to be attached to C-8 based on the HMBC correlation of the methoxy protons that appeared at δ H 4.27 with C-8 at δ C 145.04.
The isolated alkaloids (1 and 2) represent a furoquinoline base skeleton with a free C-4 proton (rarely found in nature) as elucidated from HMBC correlations ( Figure S17), other than the previously isolated furoquinoline alkaloids (usually methoxylated at C-4), in addition to the methoxy and prenyloxy groups at C-8 in compounds (1 and 2), respectively.
The isolated furoquinoline alkaloids (1 and 2) were evaluated in vitro for their cytotoxicity (S15), against HepG-2, PC-3, A-549 and MCF-7 cell lines using MTT assay in comparison with Adriamycin (Doxorubicin) as a reference compound. The obtained results revealed some sort of selectivity, which can be clarified as follow, compound (1) recorded the highest activity against HepG-2 and MCF-7 with IC 50 = 230.2 and 326.5 μM, respectively, while, it showed a moderate cell inhibition against PC3 and A549. On the other hand, compound (2) recorded slight cell inhibition against HepG-2, PC-3 and A-549, while its highest activity was recorded against MCF-7 with IC 50 = 234.2 μM.
Since furoquinoline alkaloids are classified as alkylating agents that tend to assemble covalently between the stacks of paired nucleotides in the DNA double helix, and hence lead to cell death (Wink 2007). So, activity of the isolated alkaloids in the present study may be attributed to their ability to form covalent bonds with the base pairs of the DNA (Mohammed et al. 2016), which requires the presence of hydrogen bond donors (i.e. -OH). In a former study by our group (Mohammed et al. 2016), we reported the SAR of the isolated furoquinoline alkaloids (Aegelbine-A and -B) and their cytotoxic activity. From the obtained results, we concluded that; as long as the number H-bond donors increased the activity increased, i.e. Aegelbine-A (with phenolic ortho-di-OH groups) was highly active than Aegelbine-B (compound 2 in the present study).
The presence of the four base pairs together with the unique pattern between H-bond donors and H-bond acceptors in the major groove allow greater specificity for the interaction more than in the minor groove of DNA (Rohs et al. 2010).
This fact helps to unveil the reason for the cytotoxic activity of the isolated alkaloids, even so, they both have one -OH group at C-7 (H-bond donor); compound (1) with an electron donating group (-OCH 3 at C-8) adjacent to the -OH group, helps in and stabilises the H-bond formation by rebalances the charge distribution (Li et al. 2006), while in compound (2), the presence of the bulky prenyloxy group at C-8 causes steric hindrance for the interaction with DNA base pairs, which effects on or destabilises the H-bond formation (Mohammed et al. 2016).
This comes in agreement with the previous report by Nam et al. (2005), who concluded that the presence of a single methoxy group at the C-8 position of the furoquinoline skeleton is a critical for the inhibitory activity against human PDE5A. Moreover, the presence of a methylenedioxy group (at 6,7-positions) adjacent to the C-8 methoxy group inhancing the inhibitory activity of the furoquinoline alkaloids against human carcinoma cell lines (Nouga et al. 2016). Furthermore, the presence of a substituted prenyloxy group at the C-7 position of the furoquinoline alkaloids with a free C-8 position revealed a significant activity against HeLa cell line (komala et al. 2006).

Plant materials
The whole plant A. majus was collected from the Delta region [along the agricultural road to Shebin El-kom city, https://goo.gl/maps/5RkZPiN9Bu32 (30°32′49.7″N 31°06′11.2″E)] El-Menoufia governorate, Egypt in April 2010. It was kindly identified by Mrs. Theresa Labib, Head of the Taxonomy specialists at El-Orman Botanical Garden, Giza, Egypt. A voucher specimen (No. 01/04/03) has been deposited at the Herbarium of El-Orman Botanical Garden, Giza, Egypt. The plant materials were air dried, finely powdered and used for the extraction.

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 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 F 254 (Merck) was used for analytical TLC.

Conclusion
The current study revealed the first isolation of furoquinoline-type alkaloids from A. majus beside that, these alkaloids represent a rarely ones with a free C-4 proton. The resulted cytotoxicity depends on their structure features; as long as the H-bond donor increased with an electron releasing group, the activity increased.

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

Funding
This work was Supported by National Research Centre, Cairo, Egypt; University of Southern Denmark, Odense, Denmark, for providing the facilities for instrumental analysis.