LC-ESI-MS/MS profiling of alkaloids and antiproliferative activity of Pachypoduim lamerei drake leaves

Abstract In the present study, evaluation of the antiproliferative activity of Pachypodium lamerei Drake leaves (family Apocyaceae) against human breast cancer cell lines MDA-MB-231 was done for the total methanolic extract, crude alkaloidal mixture and ursolic acid using the MTT colorimetric assay. The methanolic extract showed the strongest antiproliferative activity followed by ursolic acid and crude alkaloidal fraction with an IC50 equal to 6.2, 14.55 and 56.3 µg/ml respectively compared to oleocanthal. It is the first record for the LC/ESI-MS/MS alkaloidal profiling of the leaves of P. lamerei. Seven alkaloids were tentatively identified according to their fragmentation patterns. Four alkaloids were related to the parent indole class and two alkaloids belong to the quinoline class in addition to one steroidal alkaloid with a pregnan nucleus. Phytochemical investigation of the methanolic extract led to the isolation of three triterpenoidal compounds including ursolic acid, 11,12-didehydroursolic acid lactone and ursolic acid lactone. Graphical Abstract


Introduction
Family Apocynaceae (known as Milkweed family) is a large family containing at least 150 genera and 1700 species (Aniszewski 2007). This family is rich in many secondary metabolites including alkaloids as major component and it also contains iridoids, flavonoids, saponins (Watson and Dallwitz 1994) which may account for its diverse biological effects including antihypertensive, anticancer (Castro 2006), antibacterial (Britto et al. 2011), antimalarial effects and treatment of cardiac disorders (Aniszewski 2007). Genus Pachypodium Lindl comprises 23 species; among the most known is P. lamerei which is endemic to Madagascar (Rapanarivo et al. 1999). This specie was previously investigated for its anti-inflammatory, anti-pyretic, gastroprotective, antihyperglycemic and antimicrobial activities (El-Kashef, Hamad, Khalil, Abd-Elbaky, et al. 2015;El-Kashef, Hamed, Khalil, and Kamel 2015).
The present study adopted LC-ESI-MS/MS to tentatively identify various classes of alkaloids detected in P. lamerei Drake leaves for the first time, depending on the diagnostic ions representing alkaloids in family Apocynaceae. We were encouraged to assess the antiproliferative activity of P. lamerei Drake against human breast cancer cell lines MDA-MB-231 depending on previous studies reported by the Egyptian National Cancer Institute's (NCI) (Zawilla 2011) stating that in Egypt, breast cancer represents 18.9% of total cancer cases (35.1% in women; 2.2% in men).

Tentative alkaloidal identification
It is the first record for the alkaloidal profiling of P. lamerei leaves. In the present work seven alkaloids were tentatively identified according to their MS 2 fragmentation patterns [ Table 1].
Both compounds 1 and 7 with a pseudo molecular ion peaks appearing at [M þ 1, m/z; 283 and 415] respectively upon further MS/MS analysis showed a strong signal at [m/z; 124] and other characteristic signals at [m/z; 254,152,144,130] (Biemann et al. 1966) explained by the bond cleavage pattern seen in ( Figure S3). Upon electron impact, ring C suffers a reverse Diels-Alder fission with expultion of C-3 and C-4 to give a fragment at m/z; 254, which can conveniently break at the benzylic position to give the piperidine fragment (m/z; 124) forming the base peak. Incomplete Diels-Alder fission of ring C affords a radical-ion, which possibly cyclizes to fragment (m/z; 152). The mass spectra of 1 and 7 exhibited a remarkably similar pattern, shifted in the m/z; values of the peaks due to the different substituents present in compound 7 equivalent to a total of m/z; 44 (equal to a methyl group attached to the nitrogen atom þ a methoxy substitution on the aromatic ring) ( Figure S4) Figure S5) (Wesolowska et al. 2016), thus compound 7 was identified as desacetyl vindoline.
Compound 2, [M þ 1, m/z; 327], was tentatively identified based on NIST library as conessidine ( Figure S6). A base peak appeared at m/z; 71 which is due to the methyl amino group and carbon atoms 1, 2 and 3 (Biemann et al. 1966). In addition to the pseudomolecular ion peak [M þ 1] an intense [M þ 1-15] fragment due to the loss of a methyl group a to the nitrogen as well as a peak at m/z; 56 attributed to the nitrogen atom connected to C-20 and the three carbons associated with it. A homologous series at 56, 70 and 84 corresponding to primary, secondary and tertiary amino functions was detected; the intensity of the peaks increases in the same order (Manske 1967).
Camptothecin (3) and 10-hydroxycamptothecin (4), two quinoline alkaloids, showed two pseudomolecular ion peaks at [M þ 1, m/z; 349] and [M þ 1, m/z; 365] respectively ( Figure S7). A typical daughter ion peak appearing at [M þ 1-44, m/z; 305] which corresponds to the loss of carbon dioxide moiety (Amna et al. 1966) correspond to the base peak of compound 3. A difference by 16 Da units was seen in each and every molecular ion peaks of compound 3 and compound 4 confirming that 4 is a derivative of 3 containing an extra hydroxyl group, in addition to the appearance of a base peak at [m/z; 321] equivalent to [(M þ 1-44) þ 16, m/z; 305 þ 16] (Figures S8 and S9).The neutral losses of 28 Da forming the peaks at m/z; 277 and m/z; 249 can be assigned to ethylene and carbon monoxide, respectively (Srimany et al. 2011).
The MS 2 spectrum of compound 5 ( Figure S10) showed a pseudomolecular parent ion peak at [M þ 1, m/z; 371] with a base peak at [M þ 1, m/z; 267]. The remarkable similarity between this fragmentation pattern and that of the parent compound rhazimanine (Atta-ur-Rahman and Habib-Ur-Rehman 1986) lead to the tentative identification of compound 5 to be a hydroxyl derivative of rhazimanine with the hydroxyl substituent placed on the aromatic ring of the structure, a mass difference of 16 Da was seen in each ion peak between rhazimanine (isositsirkine) and its hydroxyl derivative as the aromatic portion of this compound remains intact during the fragmentation process. Previous report for the isolation of 11-hydroxyrhazimanine from Hunteria zeylanica (Apocynaceae) by (Xu et al. 2006) supported our finding.
As for compound 6 it appeared at [M þ 1, m/z; 409], which correspond to molecular weight of bleekerine alkaloid (408) previously separated from Bleekeria vitiensis (Kilminster et al. 1972). The maximum abundance in the tandem mass spectrum of this compound was seen for the daughter ion peak at [m/z; 242] corresponding to the molecular formula (C 14 N 2 O 2 H 13 ) þ ( Figure S11). It resembles tetrahydroalstonine in its fragmentation behavior (NIST). All characteristic ion fragments are represented in (Table 1).

Anti-proliferative activity
The results ( Figure S1) revealed that the strongest anti-proliferative activity was seen for the 80% methanolic extract with an IC 50 equal 6.2 mg/ml, lower than that of the used positive control (oleocanthal), followed by ursolic acid, isolated as pure compound from the leaves of P. lamerei with an IC 50 equal 14.55 mmol/ml. These results are in good agreement with previously reported distinct multifunctional anticancer activities of ursolic acid in the prevention and treatment of various types of cancer cells (Kim and Park 2002;Shanmugam et al. 2010). While the highest IC 50 value (56. 3 mg/ml was seen for the crude alkaloid fraction opposing the previous data on various classes of alkaloids (Lu et al. 2012). It is worth mentioning that the survival percentage of the breast cancer cells was significantly reduced in the highest tested concentration 100 mg/ml.

Conclusion
The methanol extract of P. lamerei leaves with an IC 50 less than 30 lg/ml, as recommended by the National Cancer Institute (USA), is considered cytotoxic (Suffness and Pezzuto 1990), thus further investigation of the antiproliferative activity and in vitro studies against other types of cancer cell lines is recommended.