Cytotoxic cardiac glycosides from the fruit (pods) of Adenium obesum (Forssk.) Roem. & Schult

Abstract Phytochemical investigation of methanolic extract of Adenium obesum led to the isolation of 42 (1–42) compounds belongs to cardiac glycosides, triterpenoids and steroids. The chemical structures of isolated compounds were elucidated by spectral techniques UV, IR, NMR and FAB MS. The cardiac glycosides were tested against three human cell lines, 3T3 (normal cells), HeLa (Human cervical cancer cell lines) and PC-3 (Human prostate cancer cell lines). The cardiac glycoside, honghelin (4), obeside B (5) and obeside C (6) showed significant effects against cell lines Hela, 3T3 and PC-3 compared to standard drug doxorubicin. Compounds 4, 5 and 6 exhibited very low IC50 (μM) against the PC3 human cell line. 4 and 6 also showed least IC50 against the HeLa human cell lines as compared to the standard drug doxorubicin whereas these three compounds showed effect on 3T3 cell line with high IC50 values compared to drug cycloheximide.


Introduction
Adenium obesum, commonly known as desert rose, belongs to the Apocynaceae family. It is a shrub or small tree with a height up to 6 m. Due to its attractive flower colours and swollen caudex, this species is widely produced as an ornamental plant (McBride et al. 2014 Plant stems and branches have graze pale to greyish green colour and contain a sticky and white coloured juice. Desert roses originate from Africa, south of the Sahara from Senegal to Sudan and Kenya, and through Saudi Arabia, Oman and Yemen. It is known in Africa as organic pesticide and for deliberately ending pregnancy, hepatotoxicity and treatment of sexually transmitted diseases, ear ache and tooth decay (Abalaka et al. 2013(Abalaka et al. , 2014(Abalaka et al. , 2015 as well as for its anti-lice, antibiotic, anti-lucusticidal and anti-molluscicidal (Versiani et al. 2014) andanti-oxidant (ebrahim et al. 2013) activities. Literature reports revealed that several medicinal plants produce cardiac glycosides and used as traditional medicine (el-Seedi et al. 2013). A. obesum is also a source of cardiac glycoside (cardenolides), which are potent cytotoxic compounds. Most of the cardenolides are active against different types of cancer cell lines such as MCF7, HFB4, HePG2 and HeLA, PNC1, DU145, C3H10T1/2 as well as also possesses anti-viral activity against (H1N1), influenza virus, human epidermoid carcinoma (Versiani et al. 2014).
Phytochemical investigation revealed that it contains valuable class of compounds like terpenoids, cardiac glycosides, flavonoids, anthraquinine, alkaloids, fatty acid and sugars (Versiani et al. 2014). Due to their importance in pharmaceutical value and therapeutic activities, there is a need to perform detailed phytochemical analysis of the fruit and flower of A. obesum. During our study, we isolated 42 (1-42) compounds belongs to cardiac glycosides, triterpenoids and steroids. The chemical structures of isolated compounds were elucidated by spectral techniques UV, IR, NMR, FAB MS and comparison with those of the reported compounds. The cardiac glycosides (4-6) were tested against three human cell lines, 3T3 (normal cells), HeLa (human cervical cancer cell lines) and PC-3 (human prostate cancer cell lines), which showed significant effects against these cell lines compared to the standard drug doxorubicin and cycloheximide. We report here for the first time these isolated compounds and their activities.
Honghelin (4) was obtained as a white crystalline solid which showed a peak at mz/ 533 (M + -H) corresponding to the molecular formula C 30 H 46 O 8 (M + ) in FAB Mass (-ve) mass spectrum. IR spectrum showed absorption band for hydroxyl group at 3413 cm −1 α β-unsaturated lactone ring at 1705 cm −1 with a shoulder at 1735 cm −1 and for olefinic group at 1627 cm −1 . The UV spectrum also confirmed the presence of five members α β-unsaturated lactone ring (λ-max at 216 nm). 1 H NMR completely agreed with those of the reported data of 4. Compound 5 and 6 also obtained as off-white crystalline solid having molecular formula C 30 H 46 O 9 and C 32 H 48 O 10 , respectively. The molecular formula of these compounds were deduced with the help of FAB Mass (-ve) mass spectrum which showed the (M-H) + ion peaks at 549 and 591 respectively. UV, IR and 1 H NMR data completely agree with those of reported data of 5 and 6. (Yamauchi & Abe 1990) ( Figure 1S).
Honghelin (4), obeside B (5) and obeside C (6) were explored for their cytotoxic activities against the three human cell lines, 3T3, HeLa and PC-3 which showed significant effects on these cell lines. Compounds 4, 5 and 6 exhibited very low IC 50 (μM) against the PC3 human cell line whereas honghelin (4) and obeside C (6) were also showed least IC 50 against the HeLa human cell lines compared to standard drug doxorubicin. All three compounds showed effect on 3T3 cell line with high IC 50 values compared to the used drug cycloheximide (Table 1S).
On the basis of these results, it is concluded that honghelin (4), obeside B (5) and obeside C (6) showed significant effect on cancer cells at lower dose as compare to doxorubicin.

Conclusion
Phytochemical investigation of the ethyl acetate fraction of A. obesum pods extracts from repeated standard chromatographic techniques using silica resulted in the isolation and characterisation of 42 compounds in which three compounds, honghelin (4), obeside B (5) and obeside C (6) were selected for their cytotoxic activity and these compounds were found to be most promising bioactive constituents which less disturbed normal cell line compared to standard drug doxorubicin. The present investigation of isolated cardiac glycoside from A. obesum fruit extract can be considered as potent cytotoxic agents for the medicinal purpose.