A new pregnane glycoside from Gomphocarpus fruticosus growing in Egypt

Abstract Phytochemical investigation of Gomphocarpus fruticosus (L.) Ait. of Egyptian origin afforded the new pregnane glycoside lineolon-3-O-[β-D-oleandropyranosyl-(1–4)-β-D-cymaropyranosyl-(1–4)-β-D-cymaropyranoside], along with six known compounds. The structures of the isolated compounds were elucidated on the basis of extensive spectroscopic evidences derived from 1D, 2D NMR experiments, mass spectrometry and by comparing their physical and spectroscopic data to literature. These included the triterpenoids 3β-taraxerol, 3β-taraxerol acetate and betulinic acid, which are identified for the first time in G. fruticosus and the cardenolides uzarigenin, gomphoside and calotropin.


Introduction
The former family Asclepiadaceae (now subfamily Asclepiadoideae of family Apocynaceae) is reputed for its content of cardenolides and pregnane glycosides (Hegnauer 1964). Gomphocarpus fruticosus (L.) Ait. (syn. Asclepias fruticosa L.), subfamily Asclepiadoideae of the family Apocynaceae, is a woody perennial shrub indigenous to south and tropical Africa, also known from north Africa, Arabian Peninsula and south Europe, where it is probably introduced (Goyder & Nicholas 2001). In Egypt, it grows in Nile Delta, the Mediterranean coastal region and South Sinai where it is known as herke (Muschler 1912;Tackholm 1974). G. fruticosus is used in the traditional medicine of tropical Africa to treat malaria, diabetes, asthma, bronchitis, cardiac palpitations, as a diuretic and a treatment for anthrax in cattle (Burkill 1985). In the Arabian Peninsula it is used to treat tumors, skin diseases, scabies and itching (Mothana et al. 2014). G. species are reported to contain 5α-cardenolides with doubly linked sugars and pregnane glycosides (El-Askary et al. 1993). Several cardenolides were identified in G. fruticosus plants of different geographical origins. uzarigenin, desglucouzarigenin, gomphotin, gomphotoxin and gomphacil were reported from plants growing along the black sea (chernobai & Komissarenko 1971;Komissarenko et al. 1995Komissarenko et al. , 1997. Australian plants afforded gomphoside and afroside (carman et al. 1964), while A. fruticosa L. (syn. G. fruticosus) cultivated in Japan further afforded uscharin and calactin derivatives and coroglaucigenin and corotoxigenin glycosides (Warashina & Noro 1994a). Pregnane glycosides of ikemagenin, kidjolanin (Abe et al. 1994), lineolon and isolineolon aglycones (Warashina & Noro 1994a, 1994b were also identified in this species. cardiac glycosides have been in clinical use for the treatment of heart failure for almost two centuries. Recent findings have highlighted potential new multi-therapeutic roles for compounds like digioxin, digitoxigenin, digitonin, ouabain, oleandrin, lanatoside c and proscillaridin A in various diseases. These included cancer, cystic fibrosis of lungs, stroke and heart ischaemia and neurodegenerative diseases (Prassas & Diamandis 2008). cardenolides having doubly linked sugars, commonly found in G. species, also displayed cardiotonic action in addition to other various biological activities. Gomphoside is 10 times as potent as the cardiotonic compound digoxin and 5 times as potent as ouabin (Thomas et al. 1990). calotropin is cytotoxic against various human cancer cell lines (Gupta et al. 2009); calactin is a potential anticancer agent for treating leukaemia (Lee et al. 2012) and uscharin is an active molluscicidal agent (Hussein et al. 1994). Pregnane glycosides isolated from different Asclepidaceae plants similarly displayed various biological activities including digitalis-like (Melero et al. 2000); cytotoxic (Wang et al. 2008;Liu et al. 2014); antidyslipidemic and antioxidant activities (Sethi et al. 2013); antitrypanosomal (Gurib-Fakim & Mahomoodally 2013); chondroprotective (Sanyacharernkul et al. 2009) and anti-obesity effects (Abdel-Mogib & Raghib 2013; Elsebai & Mohamed 2015). In our plan to discover cardioactive compounds from Egyptian natural resources, we reviewed the phytochemical investigation of G. fruticosus (L.) Ait. growing in Egypt. A single report for the identification of flavonoids of quercetin, kaempferol and isorhamnetin-type glycosides could be traced in the literature (Heneidak et al. 2006). So, it was deemed of interest to investigate the cardenolide and pregnane glycosides content of the plant.
Our results for G. fruticosus lies in partial agreement with those of Seiber et al. (1983), who reported that gomphoside and afroside (the 15-OH derivative of gomphoside) are typical for this species. However, tracing of recent literature revealed that afroside (but not gomphoside) is present in other Asclepias species. Afroside was not isolated in this study, nor were the thiazoline and thiazolidine cardenolides (e.g. uscharin and uscharidin) commonly occurring in Asclepias (cheung et al. 1983).Whether these cardenolides are totally absent or/present as minor constituents, is still not clear. Further investigation of the more polar fractions of the MeOH extract is in progress as TLc screening of the methanolic mother liquor (data not shown) revealed several spots giving the same colour reactions of the isolated compounds. The other isolated cardenolides viz. uzarigenin and calotropin are of wide occurrence in the genus and family. The triterpenoids isolated in this work are reported from G. fruticosus for the first time and are also common in other species of the Asclepiadaceae (Moulisha et al. 2009;Karthikeyan & Balusubramanian 2014;Maldonado et al. 2015). Betulinic acid is an exception, as it could not be traced in any other Asclepias species and hence might be considered as another hallmark for G. fruticosus.
In conclusion: this study resulted in the isolation and identification of a new pregnane glycoside, three cardenolides and three triterpenoids, previously unreported from G. fruticosus; data which warrants more pharmacological studies of the plant extracts.

Plant material
G. fruticosus (L.) Ait. was collected from South Sinai. The identity of the plant was confirmed by Professor I. Mashaly, Dept. of Botany, Faculty of Science, Mansoura university. A voucher (1504) specimen was deposited in Department of Pharmacognosy, Faculty of Pharmacy at Mansoura university.

Extraction and isolation
The air-dried powdered aerial parts of G. fruticosus (1.4 kg) were extracted with MeOH (10 L × 5) at room temperature. Removal of the solvent under reduced pressure at 45 °c gave 171.0 g (12.21%w/w) of solid residue, which was suspended in 200 ml of MeOH, diluted to 500 ml with dist. H 2 O and defatted with pet ether and then extracted with cH 2 cl 2 (1 L × 5). Evaporation of the solvents gave 41.0 g (2.93% w/w) of pet ether extract and 45.0 g (3.21%w/w) of cH 2 cl 2 extract, respectively. The cH 2 cl 2 extract was purified by passing over activated charcoal and eluted with MeOH (2 L) to yield 21.5 g of resinous solid. The partially purified cH 2 cl 2 extract was chromatographed on silica gel column (3.5 x 70 cm) eluted with mixtures of pet ether and EtOAc. Hundred millilitre fractions were collected and fractions having similar TLc pattern (using vanillin/H 2 SO 4 as spray reagent) were pooled. Fractions (1.73 g) eluted with 5% EtOAc in pet ether afforded 1 as colourless needle crystals (42.0 mg) by crystallisation from the same solvent. Further fractions eluted with 5% EtOAc in pet ether (920 mg) contained one major spot and several minor spots. They were rechromatographed on silica gel column (1.5 × 70.0 cm) and eluted with mixtures of EtOAc in pet ether to yield crude 2 (290 mg). Purification on a silica gel column (1.0 × 60.0 cm) eluted with 100% cH 2 cl 2 afforded pure 2 as colourless needle crystals (112.0 mg). Fractions (1.44 g) eluted with 10% EtOAc in pet ether contained one major spot in addition to several minor ones. It was rechromatographed on silica gel column (1.5 × 90 cm) and eluted with the same solvent system to yield crude 3 (400 mg). Purification on a silica gel column (1.0 × 70.0 cm) eluted with mixtures of cH 2 cl 2 and MeOH afforded pure 3 as colourless needle crystals (90.0 mg). Fractions (4.23 g) eluted with 40-50% EtOAc in pet ether contained several spots. A portion of which (1.0 g) was fractionated on reversed phase silica Rp-18 open column (2.5 × 40 cm) eluted with mixtures of MeOH-H 2 O and collecting 10-ml fractions. Subfractions (210.0 mg) eluted with 60% MeOH were purified on silica gel column eluted with mixtures of cH 2 cl 2 -EtOAc (50:50) containing increasing proportions of MeOH to yield pure 4 as colourless needles (42.0 mg). Another portion (1.12 g) of fractions eluted with 40-50% EtOAc in pet ether was rechromatographed on silica gel column eluted with increasing proportions of pet ether in EtOAc. Subfractions (140.0 mg) eluted with 35% pet ether in EtOAc were further purified on silica gel column eluted with 50:50 mixture of pet ether -cH 2 cl 2 containing increasing proportions of MeOH to yield 5 as a white amorphous solid (23.0 mg) in fractions eluted with 10% MeOH and 6 and 7 as crystalline solids (13.0 and 30.0 mg) in subsequent fractions eluted with 15% MeOH.