Perisomalien A, a new cytotoxic scalarane sesterterpene from the fruits of Periploca somaliensis

Abstract The CHCl3 fraction of MeOH extract of Periploca somaliensis (family Asclepiadaceae) fruits afforded a new scalarane sesterterpene, namely perisomalien A (1), along with lupeol acetate (2), β-amyrin (3), cycloart-23Z-ene-3β,25-diol (4), and β-sitosterol-3-O-β-D-glucopyranoside (5). Their chemical structures were established by various spectroscopic analyses, in addition to comparison with the formerly reported data. Moreover, the cytotoxic activity of these metabolites was assessed towards MCF-7, HepG2, and HCT-116 tumour cell lines using sulforhodamine B (SRB) assay. Compound 4 showed the most potent cytotoxic profile with IC50 9.0 µM towards MCF-7, compared to doxorubicin (IC50 0.18 µM). Also, 1 and 4 possessed the most potent effect towards HepG2 with IC50s 26.7 and 25.9 μM, respectively. In addition, all tested compounds showed cytotoxic effects with IC50 values ranging from 19.9 to 39.3 µM against HCT-116. Graphical Abstract


Introduction
Since ancient times, herbal medicines are widely used for the treatment of various diseases (Mahmood et al. 2011). Medicinal plants play a vital role in the improvement of human health through studying the biological activities of their bioactive metabolites (Youssef 2013). Family Asclepidaceae comprises about 250 genera and over 2000 species (Sinha and Mondal 2011), that distributed in the north and tropical regions, especially Africa and East Asia . The genus Periploca consists of 14 species that distributed in Asia, Europe and Africa (Verhoeven and Venter 1994). It is represented in Saudi Arabia by four major species; P. aphylla Dcne, P. visciformis Vatke, P. Brevicoronata, and P. somaliensis Browicz (Al Musayeib et al. 2016). This genus contain medicinal plants that are traditionally used in the treatment of various ailments such as autoimmune hepatitis, rheumatoid arthritis, skin and heart diseases, wounds, digestive disorders, ulcer, headache, and diabetes (Abdel-Monem et al. 2015;Iqbal et al. 2012;Mohamed et al. 2009a). These plants exhibited a wide range of pharmacological activities including, a-glucosidase inhibition, cytotoxic, antiprotozoal, antimicrobial, anti-inflammatory, cardiotonic, antimalarial, and antioxidant (Al Musayeib et al. 2016;Lu et al. 2010). Previous phytochemical studies of the different Periploca species revealed the presence of pregnane and cardiac glycosides, triterpenes, flavonoids, sterols, sterols, tannins, and saponins (Siciliano et al. 2005;Wang et al. 2007;Abdel-Monem et al. 2015;Al Musayeib et al. 2016;Liu et al. 2016;Ben nejma et al. 2017;Ali et al. 2018). However, there are limited published data on the phytochemical constituents of P. somaliensis. Only triterpenoids were isolated from the fruits of P. somaliensis (Abdel-Monem et al. 2015). Therefore, this work aimed to discover new metabolites from P. somaliensis. The chromatographic separation of the CHCl 3 fraction of MeOH extract of P. somaliensis fruits afforded a new scalarane sesterterpene, perisomalien A (1) and four known metabolites (2-5) ( Figure 1). Moreover, the cytotoxic activity of these metabolites was assessed towards various tumour cell lines using SRB assay.

Results and discussion
Compound 1 was isolated as a white amorphous powder. It had a molecular formula C 25 H 42 O 2 based on the HR-ESIMS pseudo-molecular ion peak at m/z 375.3259 (calcd for C 25 H 43 O 2 , 375.3263 [M þ H] þ ), along with 13 C and HSQC spectral data (Figures S3, S5, and S10). This formula required 5 degrees of unsaturation, one of them for a carbonyl group and the remaining four degrees suggesting the tetracyclic nature of 1. Its IR spectrum indicated the presence of hydroxyl (3439 cm À1 ), C-H aliphatic (2945 cm À1 ), and ketone carbonyl (1719 cm À1 ) functionalities (Ibrahim et al. 2012(Ibrahim et al. , 2018. The 13 C and HSQC spectra displayed 25 carbon resonances: 6 methyls, 9 methylenes, 5 methines one of them for an oxymethine (d C 78.9), and 5 quaternary carbons including one carbonyl carbon (d C 212.5) ( Figures S3 and S5). The structure and all of the 1 H and 13 C chemical shifts of 1 were elucidated by 2 D NMR spectroscopic analysis, in particular, 1 H-1 H COSY, HSQC, and HMBC experiments (Figures S1-S6), indicating a characteristic scalarane sesterterpene structure. The 1 H and 13 C NMR spectra exhibited five singlet methyl groups at d  (Rao et al. 1991;Roy et al. 2002). The HMBC cross peaks of H-25/C-17 and H-17/C-24 established the connectivity of the methyl ketone moiety at C-17. Moreover, an oxymethine signal at d H 3.20 (dd, J ¼ 11.0, 6.3 Hz, H-3), correlating to the carbon resonating at d C 78.9 in the HSQC was observed. The HMBC cross peaks of H-1, H-19, and H-20 to C-3 established the position of the oxymethine at C-3. Three methine proton signals at d H 0.72 (dd, J ¼ 11.9, 2.6 Hz, H-5), 1.28 (m, H-9), 1.91 (m, H-14), and 2.58 (ddd, J ¼ 13.8, 11.1, 10.6 Hz, H-17), which correlated with the carbon signals at d C 55.9 (C-5), 50.7 (C-9), 45.2 (C-14), and 54.3 (C-17), respectively in HSQC spectrum were observed. Their assignment was established based on the observed correlation in the 1 H-1 H COSY and HMBC spectra ( Figure S4, S6 and S7). Analysis of the NMR data revealed that 1 was a 25-norscalarane derivative. Based on the literature, this class of metabolites was isolated commonly from the marine organisms; here is the first report for its isolation from a plant source (Gonz alez 2010). The relative configurations at all asymmetric centers were deduced from the comparison of the coupling constant values and 1 H and 13 C NMR data with those of related metabolites and further secured by the NOESY experiment. The a-configuration of H-3 was assigned on the basis of its coupling constant (J 3,2 ¼ 11.0 and 6.3 Hz) and further secured by its cross peaks with the  Figures  S7 and S9). On the basis of the above findings, 1 was identified as 3-b-hydroxy-24methyl-24-oxo-25-nor-scalarane and named perisomalien A. The known compounds were identified through the analysis of the spectroscopic data (1D, 2D NMR, and MS) and comparison of their data with those in the literature as lupeol acetate (2) (Jamal et al. 2008;Al Musayeib et al. 2013), b-amyrin (3) (Khedr et al. 2016), cycloart-23Z-ene-3b,25-diol (4) (Corsaro et al.1994), and b-sitosterol-3-Ob-D-glucopyranoside (5) (Mohamed et al. 2009b).
Compound 4 showed the most potent cytotoxic profile towards MCF-7 with IC 50 9.0 mM in comparison to doxorubicin (IC 50 0.18 mM), whereas 1 and 5 had moderate effect with IC 50 19.2 mM. On the other hand, 3 exhibited a slightly lower effect with IC 50 21.1 lM. Also, 1 and 4 possessed the most potent effect towards HepG2 with IC 50 s 26.7 and 25.9 lM, respectively. While 2, 3, and 5 possessed a weak cytotoxicity with IC 50 s 29.9, 31.7, and 33.6 lM, respectively. In addition, the tested compounds showed cytotoxic effects with IC 50 values ranged from 19.9 to 39.3 mM against HCT-116. Meanwhile, 4 was highly effective among all other compounds with IC 50 of 19.9 mM against HCT-116. It is noteworthy that 4 possessed a high cytotoxic effect towards all cancer cell lines with IC 50 values ranged from 9.0 to 25.9 mM, while 5 showed a low cytotoxic effect with IC 50 values ranging from 19.2 to 39.3 mM (Table S1; Figures S11 and S12).

Experimental
Detailed general experimental procedures, plant material, extraction and isolation, cell culture, and cytotoxicity assessment are available as Supplementary Material.

Conclusion
A new scalarane sesterterpene (1) and four known compounds (2-5) were isolated from the CHCl 3 fraction of MeOH extract of P. somaliensis and their chemical structure was identified using various spectroscopic tools. These compounds showed variable cytotoxic activity in different cancer cell lines. Compounds 1 and 4 had the most potent cytotoxic effect.

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