New Polyprenylated Phloroglucinol and Other Compounds Isolated from the Fruits of Clusia nemorosa (Clusiaceae)

Clusia nemorosa has been widely used in folk medicine to treat various ailments, including headaches and inflammation. Investigation of the fruits of Clusia nemorosa (Clusiaceae) led to the isolation and characterization of a new phloroglucinol derivative, named 6S,8S,28S-nemorosic acid (1), together with seven known compounds: friedelin (2), β-sitosterol (3), stigmasterol (4), β-sitosterol glycoside (5), kaempferol (6), quercetin (7) and dimethyl citrate (8). The structures were determined by extensive 1D- and 2D-NMR, CD and MS spectroscopic analyses.


Introduction
The Clusiaceae family, also known Guttiferae, is characterized by the presence of flower resin and latex in most of its species [1]. The genus Clusia presents a notable metabolite diversity, including benzophenones, xanthones and biflavonoids [2][3][4][5]. Several species are used in folk medicine all around the world to treat rheumatism, stomach problems and as a purgative [6]. Phytochemical and pharmacological studies of plants belonging to this genus have reported many biological activities, such as antibacterial, antioxidant, antitumour, antimicrobial and anti-inflammatory properties [7]. (-)-Nemorosonol, a prenylated OPEN ACCESS benzophenone, exhibited antimicrobial activity against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Aspergillus niger, Trichophyton mentagrophytes and Candida albicans [8]. The biflavonoids GB1-7′′O-glucoside and GB1a-7′′O-glucoside showed promising free radical scavenging capacity in in vitro assays [5]. Phloroglucinol derivatives exhibited chemopreventive properties and inhibited NO production [9]. The Clusia genus thus represents an important source of bioactive compounds.
Clusia nemorosa Mey., popularly known as "pororoca", is a tree that is widespread in the northeast region of Brazil [10]. This species has been widely used in folk medicine to treat headaches and inflammation [11]. Prior phytochemical studies of the fruits of this species led to the isolation of a polyisoprenylated benzophenone and phloroglucinol derivatives [12,13]. The additional study of this species presented herein allowed us to identify a new phloroglucinol derivative, named nemorosic acid (1), as well as seven known compounds that were identified for the first time in the fruits: friedelin (2), β-sitosterol (3), stigmasterol (4), β-sitosterol glycoside (5), kaempferol (6), quercetin (7) and dimethyl citrate (8).

General Procedures
The melting points were determined with a Mel-temp II apparatus and were uncorrected. IR spectra were obtained with KBr discs and NaCl film using a Vertex-70 spectrophotometer (Bruker Corporation, Billerica, MA, USA). UV spectra were obtained on a Shimadzu UV-mini 1240 UV-VIS spectrophotometer (Quioto, Kansai, Japan). 1

Extraction and Isolation
The dried fruits (130.0 g) were extracted with dichloromethane (2.0 L), followed by extraction with methanol (2.0 L) at room temperature three times. The two extracts were separately concentrated to give 15.0 g of CH2Cl2 extract and 27.0 g of MeOH extract. Part of the dichloromethane extract (11.0 g) was subjected to column chromatography on silica gel with dichloromethane, ethyl acetate (EtOAc) and methanol (MeOH) as eluents to give three fractions (A1-A3). The A2 fraction (3.0 g) was subjected to repeated silica gel chromatography using hexane-CH2Cl2, CH2Cl2-AcOEt and EtOAc-MeOH to furnish the compounds 1 (35.0 mg) and 5 (65.0 mg). The A1 fraction (5.1 g) was further fractionated through silica gel column chromatography using hexane-EtOAc and EtOAc-MeOH to furnish compounds 2 (20.0 mg) and the mixture of 3 and 4 (10.0 mg). Meanwhile, the dry MeOH extract (20.0 g) was suspended in H2O and was partitioned with chloroform (3 × 200 mL), ethyl acetate (3 × 200 mL) and n-butanol (3 × 200 mL). The EtOAc fraction (3.5 g) was subjected to Sephadex LH-20 column chromatography and eluted with MeOH to give five fractions. Fraction 4 was rechromatographed on Sephadex LH-20 and eluted with MeOH to give 6 (5.0 mg) and 7 (5.0 mg). Fraction 5 was rechromatographed over a silica gel column using CHCl3-MeOH (0%-100% methanol) as eluents to give 8 (35.0 mg).

Conclusions
Eight compounds, including the novel compound 1, were isolated from the fruits of C. nemorosa. The discovery of this new compound from the genus Clusia provides more spectroscopic data to characterize the components of its isolates, as well as contribute to the understanding of the taxonomy and evolution of the genus Clusia.