Brasiliensic and isobrasiliensic acids: isolation from Calophyllum brasiliense Cambess. and anti-Helicobacter pylori activity

Abstract The occurrence of chromanone derivatives has been noticed as a distinctive feature of the genus Calophyllum (Calophyllaceae). Previous studies have demonstrated that the extract of the stem bark of Calophyllum brasiliense and its chromanone-rich fractions show anti-ulcer activity in murine gastric ulcer models. In this work, brasiliensic and isobrasiliensic acids, the two main compounds of the n-hexane extract of the stem bark extract of C. brasiliense, were isolated by flash chromatography using silica gel impregnated with silver nitrate and their structures were elucidated by NMR techniques and mass spectrometry. 13C NMR data is available for the first time for both compounds. Brasiliensic and isobrasiliensic acids showed good in vitro bacteriostatic activity against Helicobacter pylori, and are responsible, at least in part, for the bacteriostatic anti-H. pylori activity of the n-hexane extract of the stem bark of C. brasiliense.


Introduction
The genus Calophyllum (Calophyllaceae) comprises ca. 200 species with a pantropical pattern of distribution. From the phytochemical point of view, the genus is distinguished for the presence of xanthones, coumarins and biflavonoids (Su et al. 2008;Filho et al. 2009). Additionally, the occurrence of chromanone derivatives has been noticed as a distinctive feature of the genus (Wang et al. 2011;Dieu et al. 2012).
Recent studies have demonstrated the presence of coumarins in the leaves of Calophyllum apetalum and stem bark of Calophyllum hosei (Joshi et al. 2013;Daud et al. 2014). Xanthones from Calophyllum inophyllum, Calophyllum soulattri and Calophyllum thorelli showed in vitro antioxidant and anti-proliferative activities (Nguyen et al. 2013;Mah et al. 2015). A new chromanone acid from Calophyllum teysmannii showed inhibitory activity against HeLa cancer cells (Lim et al. 2015) and leaves extract from Calophyllum brasiliense possesses wound healing properties (Lordani et al. 2015).
C. brasiliense is one of the most abundant species of the genus that occurs from Mexico to Brazil in periodically or permanently flooded areas (Lorenzi 1992). In Brazilian traditional medicine, the stem bark infusion and bark oleoresin of C. brasiliense ('guanandi' , 'jacareúba' , 'olandi' among others) are used for the treatment of inflammation, pain, varices, haemorrhoids, rheumatism and chronic ulcer, among others (Filho et al. 2009).
Pharmacological investigations of the stem bark of C. brasiliense demonstrated that its crude n-hexane extract and fractions display gastroprotective effect in murine models of gastric ulcer (Sartori et al. 1999;Lemos et al. 2012) and a pronounced anti-Helicobacter pylori activity (Souza et al. 2009). Additional studies pointed out that the observed antiulcer and anti-H. pylori activity of C. brasiliense could be attributed to the enriched fraction composed mainly of a mixture of chromanone derivatives (Lemos et al. 2012).
H. pylori, a known causative agent of gastroduodenal ulcers and gastric cancer, infects more than 50% of the world population. The combination of antibiotics with proton pump inhibitor has been used as a consensus treatment with efficacy. However, the emergency of antibiotic-resistant strains of H. pylori has made it a necessity the search for new effective antibiotics (Garza-González et al. 2014).
Based on our previous results of anti-H. pylori activity of a chromanones-enriched fraction of HeCb, we aimed to isolate, identify and test these compounds against the bacterium. Our efforts to isolate the main compounds of this lipophilic fraction were initially fruitless, even though preliminary HPLC, MS and NMR analyses revealed that this fraction is composed mainly of two isomeric chromanones, namely brasiliensic acid (1) and isobrasiliensic (2) acids (Lemos et al. 2012).
Despite the fact that both (1) and (2) are known and have been previously described, the isolation of these two isomeric compounds has proved to be a challenging task. They were previously obtained only as synthetic derivatives (Stout et al. 1968;Caneppele et al. 2008). By following the short description of Stout et al. (1968), our attempts to separate both acids were without success. Therefore, the present communication shows the results of the isolation of (1) and (2) (Figure 1) by column chromatography on silica gel impregnated with silver nitrate (De Vries 1964;Williams & Mander 2001) and the evaluation of their in vitro anti-H. pylori activity.

Results and discussion
(1) and (2) were reported for the first time by Stout et al. (1968) and were obtained from the oleoresin of C. brasiliense. Their structures were later corrected (Stout & Breek 1970). Caneppele et al. (2008) isolated (1) as O-methoxy-methyl ester derivative. In the present work, we report for the first time a detailed procedure for the reproducible isolation of compounds 1 and 2 with no derivatisation process using three chromatographic techniques in the following sequence: vacuum liquid chromatography, semi-preparative HPLC and flash column chromatography on silica gel impregnated with silver nitrate (for detailed information, see section 3.3) (De Vries 1964;Williams & Mander 2001). We highlight the importance of using silica gel impregnated with silver nitrate for efficient separation of the diastereoisomers. All the procedures were repeated several times in order to obtain a suitable amount of compounds for the bioassays. These procedures are quite simple, showed good reproducibility and therefore can be used for further isolation.
(1) and (2) were obtained as yellow gums. The MS data of compounds 1 and 2 are in agreement with the molecular formula of C 32 H 46 O 6 . The NMR data are reported in the Table  S1. Stout et al. (1968) and Stout and Breek (1970) barely described the 1 H NMR data, while Caneppele et al. (2008) reported 1 H and 13 C NMR data of O-methoxy-methyl ester derivatives. The 1 H and 13 C NMR data of compounds 1 and 2 displayed many shared features that are also in agreement with the data reported by Caneppele et al. (2008) for their O-methoxy-methyl ester derivatives. Some key differences between 1 and 2 can be observed in the 1 H NMR spectra ( Figure S1) that include the observance of a characteristic pseudo-triplet signal in the 1 H NMR spectra of 2 corresponding to a second prenyl side chain that is only present in isobrasiliensic acid (2).
Recently, Lemos et al. (2012) reported the anti-H. pylori activity of an enriched mixture of chromanone derivatives, obtained from the n-hexane extract of C. brasiliense stem bark. Preliminary HPLC, MS and NMR analyses carried out by Lemos et al. (2012) suggested the presence of (1) and (2). In the present work, we evaluated whether the observed activity can be attributed to these constituents isolated from the same accession of the stem bark of C. brasiliense. Both chromanones (1) (MIC = 50 μg/mL) and (2) (MIC = 12.5 μg/mL) displayed lower MIC values than that observed for the n-hexane extract HeCb (MIC = 100 μg/mL), therefore indicating that the purification process increased the antibacterial activity. Thus, HeCb, (1) and (2) can be considered as having good activity, similar the standard drug clarithromycin (MIC = 6.25 μg/mL), as shown in Figure 2. These results indicate that both chromanones play an important role in the anti-H. pylori activity of the HeCb, most notably the isobrasiliensic acid. Additionally, no MBC was recorded for compounds 1 and 2 and the HeCb, within the range of concentrations tested, indicating that the HeCb and both (1) and (2) show bacteriostatic activity under the conditions used in our in vitro tests. Recent studies postulate that bacteriostatic drugs may have bactericidal effects under certain conditions. Bactericidal and bacteriostatic activities of a drug can be influenced, in vitro, by bacterial growth conditions, bacterial density, test duration and extent of reduction in bacterial numbers (Pankey & Sabath 2004) and clinically, by host, drugs and pathogens (Nemeth et al. 2015).

Conclusions
In summary, it is possible to conclude that brasiliensic and isobrasiliensic acids are responsible, at least in part, for the bacteriostatic activity of HeCb against anti-H. pylori and that isobrasiliensic acid (2) was more active than brasiliensic acid (1). Both chromanones may be considered as potential phytomedicines in the treatment of ailments whose principal cause is related to H. pylori.