Triterpene Esters: Natural Products from Dorstenia arifolia (Moraceae)

The phytochemical study of Dorstenia arifolia Lam. (Moraceae) has led to the identification of 18 triterpenes esterified by fatty acids, five triterpenes without esterification, 12 triterpenes esterified by acetic acid, together with a known furanocoumarin: α-amyrin (1), β-amyrin (2) α-amyrin acetate (3) β-amyrin acetate (4), α-amyrin octanoate (5), β-amyrin octanoate (6), α-amyrin decanoate (7), β-amyrin decanoate (8), α-amyrin dodecanoate (9), β-amyrin dodecanoate (10), α-amyrin tetradecanoate (11), β-amyrin tetradecanoate (12), α-amyrin hexadecanoate (13), β-amyrin hexadecanoate (14), glutinol (15), glutinyl acetate (16), 11-oxo-α-amyrin (17), 11-oxo-β-amyrin (18), 11-oxo-α-amyrin acetate (19), 11-oxo-β-amyrin acetate (20) 11-oxo-α-amyrin octanoate (21) 11-oxo-β-amyrin octanoate (22), 11-oxo-α-amyrin decanoate (23), 11-oxo-β-amyrin decanoate (24) 11-oxo-α-amyrin dodecanoate (25) 11-oxo-β-amyrin dodecanoate (26), ursa-9(11),12-dien-3-yl acetate (27), oleana-9(11),12-dien-3-yl acetate (28), ursa-9(11),12-dien-3-yl decanoate (29), oleana-9(11),12-dien-3-yl decanoate (30), 12,13-epoxyolean-3-yl acetate (31), 12,13-epoxyolean-9(11)en-3-yl acetate (32), taraxeryl acetate (33), lupenyl acetate (34), lanosta-8,24-dien-3-yl acetate (35) and psoralen (36). The identification of the triterpene compounds isolated as isomeric mixtures obtained from the hexane extract was based mainly in mass spectra and 13C-NMR data. The long-chain alkanoic acid esters of the triterpenes α- and β-amyrin; 11-oxo-α- and 11-oxo-β-amyrin; ursa- and olean-9(11),12-dien-3-yl; have not been reported before in the literature as constituents of the Dorstenia genus.

Triterpenes are a class of natural products found especially in plants. The triterpene acids exhibit important biological and pharmacological activities, including anti-inflammatory, antimicrobial, antiviral, cytotoxic and cardiovascular effects [9]. The compounds α-amyrin and β-amyrin, commonly found in medicinal plants, have many bio-active properties. Some studies have demonstrated that the α/β amyrin triterpene mixture also has many biological functions, including analgesic, antimicrobial, anti-inflammatory properties [10].
Some Dorstenia species show the strong ethnobotanical indications concerning anti-snake bite poisoning properties. Such effects may be related to the presence of triterpenoids [11]. The presence of triterpenes esterified by fatty acids has been a common characteristic in plant species from Brazilian Restinga [12] mainly in the Erythroxylaceae [13].
In Brazil, pharmacological information about the Dorstenia genus are very few [14]. No previous phytochemical study on Dorstenia arifolia Lam. has been reported. This paper deals with the isolation and the structural elucidation of 18 long-chain alkanoic acid esters of some triterpene skeletons, five triterpenes, 12 triterpenes esterified by acetic acid and only one already known furanocoumarin. The present study has focused on the analysis of terpenoidal compounds from Dorstenia arifolia, using phytochemical methodology. This study may be an excellent tool to show the value of classical phytochemical analysis procedures based on chromatographic isolation combined with spectroscopic identification, for the analysis of low-polarity plant extracts [11].

Results and Discussion
Powdered leaves and rhizomes of D. arifolia were successively extracted with n-hexane. The extracts were submitted to repeated column chromatography to afford various pentacyclic triterpenes esterified by fatty acid and a coumarin. The 1 H-and 13 C-NMR as well as the MS of the isolated compounds were consistent with the literature records.
The chemical constituents of the genus Dorstenia have been reported to be coumarins/ furanocoumarins, flavonoids, triterpenoids and triterpenoid esters [15]. This is the first time that long-chain alkanoic acid serial esters (Figures 1 and 2) have been isolated from the Dorstenia genus and identified as isomeric pairs.     Most of the triterpenes found belong to the oleanene/ursene series, characterized by a base peak at m/z 218. Unequivocal differentiation between α-and β-amyrin (1, 2) could be seen by examination of the relative intensities of the peaks at m/z 189 and 203: β-amyrin (2) has a m/z 203 peak around twice the intensity of the m/z 189 peak, while α-amyrin (1) spectra shows both peaks with similar intensities. The triterpenes of the 11-oxo-α-amyrin (17) and 11-oxo-β-amyrin (18) types present as characteristic signals m/z 232, m/z 273 and m/z 135, the latter being quite abundant. Taraxeryl acetate (33) was identified only by MS, mainly due to the base peak at m/z 204, which originates from rings D and E of an D 14 -taraxerene. Another important peak is at m/z 344, which originates from a retro Diels-Alder decomposition with ring-D opening and confirms both the unsaturation and the presence of an acetoxy group at C-3. The most important feature at glutinyl acetate (16) being the base peak at m/z 274, followed by a peak at m/z 259 (274-Me), which characterizes D 5 -unsaturated skeletal (Table 1) [16].

Plant Material
Samples of D. arifolia Lam. (Moraceae) were collected in Rio de Janeiro, Brazil. The botanical identification was provided by Dr. Marcelo Dias Machado Vianna Filho and a voucher specimen (RB 517081) was deposited in the Herbarium of the Jardim Botânico do Rio de Janeiro, Rio de Janeiro, Brazil.
Fractions D (796 mg) and E (287 mg) were eluted on a silica gel column chromatography with hexane/ethyl acetate 3% and submitted to a new silica gel column chromatography to yield ( Figure 1): α-amyrin (1).
Powdered rhizomes (10 g) of D. arifolia were exhaustively extracted with n-hexane. The solvent was removed under reduced pressure to yield the hexane extract (DaEHR). DaEHR was chromatographed over silica gel (0.063-0.200 mm, Merck) using hexane-ethyl acetate of increasing polarity, which yielded mixed fractions G, H, I, J, K, L, M and N.
Fraction N (135 mg) was eluted on a silica gel column chromatography with hexane/ethyl acetate 15% and was submitted to a small column chromatography using Sephadex LH-20 and CHCl 3 /MeOH (1:1) as eluent to yield a furanocoumarin (1 mg) ( Figure 2). All compounds were identified by interpretation of the results of the spectra and comparison with literature data.

Chromatographic Analysis
GC-MS analysis was performed by using a GC-MS QP5000 Shimadzu, with electron impact ionization (70 eV). The column used was a DB-5MS (30 m × 0.25 mm × 0.25 µm) with injector temperature at 290 °C and GC-MS interface temperature at 250 °C. Column temperature was programmed from 100 °C at 320 °C (held during 120 min), ranging 10 °C/min. Helio was used as carrier gas. The mixtures A-N were analyzed by GC-MS which furnished a fast differentiation among important skeletons. The NMR data were only used to confirm the results proposed by mass spectra.

Basic Hydrolysis of Triterpene Ester Derivatives
Some triterpene ester derivatives (compounds 7-12 and 22-26) were submitted to hydrolysis by adding 4 mL of a solution of NaOH in MeOH 0.5 N to 100 mg of mixture for 10 hours. After this time, the reaction medium was saturated with NaCl 360 g/L and the triterpenes were extracted with CHCl 3 . The aqueous solution was acidified with 4 mL of HCl 0.5 N followed by the extraction with CHCl 3 . This resultant organic phase was washed and dried over Na 2 SO 4 , yielding the fatty acids.

Conclusions
GC-MS has proved to be a very powerful tool affording both the separation and the individual characterization of terpenoidal isomers which could not to be separated by conventional PLC procedures. MS data furnish a fast differentiation among important skeleton types, some of them with potential biological interest shown in literature.
Pentacyclic triterpenes and a furocoumarin from Dostenia arifolia were identified. These compounds may be related to the folk utilization of Dorstenia species as antiophidicals. The utilization of Dorstenia plants as antiophidicals may be inferred to be both due to a venom-inactivating action and to the analgesic and antiinflamatory properties of the various triterpenes [11].