Steroids and Triterpenoids from Corypha taliera Roxb: A Critically Endangered Palm Species of Bangladesh

Corypha taliera Roxb. is a critically endangered palm species. The tree grown in the campus of the University of Dhaka, Bangladesh used to be considered as the only living species in the world. This palm tree raised some interests due to several bioactivities observed earlier and later systematic scientific studies have been undertaken to explore for its secondary metabolites. In the present study, a thorough phytochemical investigation was conducted to explore its chemical features. Initially, the unripe fruits of this palm was extracted with methanol. Later on, it was fractioned by the modified Kupchan partitioning method in to n-hexane, carbontetrachloride, chloroform and aqueous soluble fractions. The n-hexane soluble fraction was subjected to Column Chromatography (CC) over Sephadex LH-20 and the column was eluted with n-hexane-dichloromethane-methanol (2:5:1) mixtures. From the collected fractions stigmasterol (1), β-sitosterol (2), β-amyrin (3), lupeol (4) and betulinic acid (5) were isolated by Preparative Thin Layer Chromatography (PTLC). The structures of these purified compounds were established by extensive spectroscopic analysis and by comparison of their spectral data with published values as well as co-TLC with authentic samples. This is the first report of isolation of steroids and terpenoids from this plant.

INTRODUCTION

Palm trees are renowned for their contribution to the human race. They can provide food and arrange landscaping for pleasing appearance. Medicinal scientists also put some light on their chemical and biological aspects for treating various pathological states (Asase et al., 2010; De Smet, 2002; Agbabiaka et al., 2009). Many palm species are now in a very challenging and endangered condition for their existence. In Bangladesh, C. taliera is considered as a very rare species and the only one found in the campus of University of Dhaka, Bangladesh. The tree was first discovered in 1919 in the then Bengal. The only record of C. taliera was reported in the University of Dhaka, Bangladesh. In the absence of any other record, the solitary tree was considered the last living species in the World (Johnson, 1998; Chowdhury et al., 2010).

C. taliera (Bengali name-tali palm; Family:Palmae) is a giant monocarpic palm that grows very slowly upto 10 m in height and 70 cm in diameter near the base. It is thought to be distributed in Bangladesh, India and throughout the tropical Asia and takes close to a century for flower to blossom, which will ultimately lead to gradual death. As a part of the continuous studies on medicinal plants of Bangladesh (Begum et al., 2010; Rahman et al., 2011), the preliminary antimicrobial, antioxidant and cytotoxic activities of the unripe fruits of this palm was reported earlier (Chowdhury et al., 2010). No other biological or chemical work has been conducted on this plant. In the present manuscript, the isolation stigmasterol (1), β-sitosterol (2), β-amyrin (3), lupeol (4) and betulinic acid (5) was reported for the first time from this palm species.

MATERIALS AND METHODS

General experimental procedures: Gel permeation chromatography was performed on Sephadex LH-20 while column chromatographic separation was achieved over silica gel (mesh 70-230). The ¹H NMR spectra were recorded using a Bruker AMX-400 (400 MHZ) instrument in CDCl₃ and the δ-values for ¹H and ¹³C data were referenced to the residual non-deuterated solvent signals. All solvents were of analytical grade.

Collection of plant materials: The fruits of C. taliera were collected from the campus of the University of Dhaka, Bangladesh in the month of April, 2009. A voucher specimen (accession No. DACB-34180) for this collection has been deposited in Bangladesh National Herbarium, Dhaka.

Extraction and isolation of compounds: The unripe sun dried and powdered fruits (500 g) of C. taliera was soaked in 1.5 L methanol for 10 days with occasional shaking and stirring and filtered through a cotton plug followed by Whatman filter paper No. 1. The extract was then concentrated by using a rotary evaporator at reduced temperature and pressure. A portion (5 g) of the concentrated methanol extract was fractioned by the modified Kupchan partitioning method (VanWagenen et al., 1993; Anjum et al., 2013). Evaporation of solvents yielded n-hexane (1.25 g), carbon tetrachloride (0.55 g), dichloromethane (0.60 g) and aqueous soluble (2.00 g) materials. An aliquot (1.10 g) of the n-hexane soluble fraction was subjected to Column Chromatography (CC) over Sephadex LH-20 and the column was eluted with n-hexane-dichloromethane-methanol (2:5:1) mixtures to give a total of 40 fractions, each 5 mL.

Compound 1 was obtained as colorless crystalline mass from fractions 6 and 7. Compound 2 was isolated as white amorphous powder from fraction 9 by preparative thin layer chromatography (PTLC, stationary phase silica gel F₂₅₄, mobile phase-10% ethyl acetate in toluene, thickness of plates-0.5 mm). Similar PTLC of fractions 10-12 and 17 over silica gel F₂₅₄ using 15% and 20% ethyl acetate in toluene afforded compound 3 (10 mg) and 4 (8 mg), respectively as white amorphous mass. On the other hand, compound 5 was isolated as white gum from fractions 21-22 by PTLC over silica gel (F₂₅₄) using 30% ethyl acetate in toluene as the developing solvent.

RESULTS AND DISCUSSION

The ¹H NMR spectra of compounds 1 and 2 (Table 1, Fig. 1a) readily demonstrated the steroidal nature of these compounds and were superimposable to the ¹H NMR spectra previously recorded for stigmasterol and β-sitosterol (Jahan et al., 2010) from the same laboratory. The identity of the compounds was further confirmed by co-TLC with authentic samples.

Fig. 1(a-c):	Structure of the isolated compounds (a) 1 and 2 represent stigmasterol and β-sitosterol, respectively, (b) 3 represents α-amyrin and (c) 4 and 5 represent lupeol and betulinic acid, respectively

Table 1:	1H NMR (400 MHZ, CDCl3) data of compounds 1-5
δ (ppm): Chemical shift expressed in parts per million, J in Hz: Coupling constant expressed in Hertz, mult: Multiplet, s: Singlet, br.s: Broad singlet, d: Doublet, dd: Double doublet, t: Triplet

The ¹H NMR spectrum (400 MHZ, CDCl₃) of compound 3 (Table 1, Fig. 1b) revealed eight singlets at δ 0.95 (H₃-28), 1.00 (H₃-29, H₃-30), 0.79 (H₃-24), 0.87 (H₃-23), 0.85 (H₃-25), 1.04 (H₃-26) and 1.07 (H₃-27). Integration of the singlets demonstrated the intensity for three protons. It suggested the presence of eight methyl groups in the molecule. It also revealed a triplet (J = 3.2 Hz) of one proton intensity centered at δ 5.12, which could be assigned to the olefinic proton (H-12). The double doublet (J = 11.0, 5.0 Hz) centered at δ 3.23 could be ascribed to the oxymethine proton at C-3. The splitting pattern of this proton suggested the beta orientation of the hydroxyl group. The above spectral features are in close agreement to those observed for α-amyrin (Parvin et al., 2009). Again the identity of this compound was substantiated by co-TLC with authentic sample.

The ¹H NMR spectrum (400 MHZ, CDCl₃) of compound 4 (Table 1, Fig. 1c) showed a double doublet (J = 11.5, 5.02 Hz) of one proton intensity at δ 3.20 typical for H-3 of a triterpene type carbon skeleton. The broad singlets at δ 4.67 and 4.55 (1H each) were assigned to the vinylic protons at C-29. A multiplet of one proton intensity at δ 2.28 was attributed to H-19. The spectrum also displayed six singlets at δ 0.78, 0.82, 0.84, 0.93, 0.95, 1.02 of three proton intensity. A broad singlet of three proton was also appeared at δ 1.67. All of these resonances are assignable to the methyl group protons at C-4 (H₃-24), C-17 (H₃-28), C-10 (H₃-25), C-14 (H₃-27), C-4 (H₃-23), C-8 (H₃-26) and C-20 (H₃-30), respectively. By comparing these with published ¹H NMR data for lupeol as well as by co-TLC with authentic sample established its identity as lupeol (4) (Sultana et al., 2010).

The ¹H NMR spectrum of compound 5 (Table 1, Fig. 1c) revealed the presence of a lupene type carbon skeleton. It displayed signals attributable to an exomethylene group at δ 4.60 and 4.73 (1H, each, br. s), which together with an allylic methyl at δ 1.68 indicated an isopropenyl functionality. The double doublet centered at δ 3.17 could be assigned to H-3. The large couplings (11.2 and 5.2 Hz) of this H-3 with the vicinal methylene protons suggested the β (beta) orientation of the hydroxyl group at C-3. In addition, the spectrum also showed a multiplet at δ 2.85 for the methine proton at C-19 and five methyl group resonances as singlets at 0.74 (H₃-24), 0.81, 0.93 (H₃-23), 0.96 (H₃-26) and 0.97 (H₃-27). On this basis, compound 5 (Fig. 1) was characterized as betulinic acid. The identity of 5 as betulinic acid was confirmed by comparison with published values (Parvin et al., 2009) as well as by co-TLC with an authentic sample.

The present study explored the presence of steroids and triterpenes from C. taliera. Among the isolated compounds, betulinic acid and lupeol have previously been reported for anticancer activity (Patocka, 2003; Saleem, 2009). It might have connection with the cytotoxicity exhibited in brine shrimp lethality bioassay by C. taliera fruits (Chowdhury et al., 2010). Preliminary TLC screenings revealed that the extractives of C. taliera fruits might have more bioactive triterpenes. Further bioactivity guided isolation is required to identify the bioactive principles.

CONCLUSION

The unripe fruits of C. taliera were subjected to phytochemical investigation, which yielded steroids and triterpenes stigmasterol (1), β-sitosterol (2), β-amyrin (3), lupeol (4) and betulinic acid (5) for the first time from this palm tree. Among these lupeol and betulinic acid are renowned for having anticancer activities. More extensive phytochemical and biological investigations are required to isolate the potential bioactive components from this plant species.