Phytochemical investigation of Erythroxylum rimosum O. E. Schulz

Erythroxylum rimosum O. E. Schulz is a species restricted to the northeastern region of Brazil, found in the states of Ceará, Piauí, Sergipe and Bahia, occurring respectively in Restinga, Cerrado and Carrasco vegetation. The study of the extract of E. rimosum, reported the identification of pentacyclic triterpenes, steroid, tropic alkaloid and flavonoids. Thus, a chromatographic study of its crude ethanol extract was carried out. The botanical material of the aerial parts was collected in the municipality of Pirambu, state of Sergipe and identified by Profa. Dra. Ana Paula do Nascimento Prata, Department of Biology, Federal University of Sergipe (FUS). It was then oven dried with circulating air at an average temperature of 40 ° C, ground in a mechanical mill and subjected to steeping with 95% EtOH. The BSE (105 g) was dissolved in a methanol: water (7:3 v/v) solution and partitioned with the following solvents: hexane, dichloromethane and ethyl acetate. The AcOEt phase was subjected to column chromatography, using silica gel 60 as stationary phase and as mobile phase, the Hex, AcOEt and MeOH solvents, pure and in binary mixtures in increasing order of polarity. This yields 30 fractions which after analytical thin layer chromatography (TLC) were pooled according to their respective retention factors (Rfs). The fractions from 23 to 25 were submitted to High Performance Liquid Chromatography Coupled to a Diode Array Detector (HPLC-DAD). Getting yourself 8 fractions. From fractions 4 and 2 the coded substances of Er-1 and Er-2 respectively were obtained. They have had their structures identified by 1H-NMR, 13C, and twodimensional techniques in comparison with literature data, namely: kaempferol-3-rutinoside and quercetin-3-O-β-D-glucopyranoside-αL-raminoside, two glycosylated flavonoids that are being reported for the first time in the study species.


Introduction
E. rimosum O. E. Schulz is a species restricted to the northeastern region of Brazil, found in the states of Ceará, Piauí, Sergipe and Bahia, occurring respectively in Restinga, Cerrado and Carrasco vegetation. 1 The study of the crude ethanolic extract of E. Rimosum leaves carried out by RIBEIRO, (2011), reported the identification of pentacyclic triterpenes (α-amirin, β-amirin), steroid (β-sitosterol), tropic alkaloid and flavonoids. 2

Results and Discussion
The substance encoded as Er-1 was isolated as a brown solid with 20 mg. The ¹³C-APT spectrum obtained at 100 MHz in CD 3 OD showed the presence of 27 signals. Of these 9 were attributed to nonhydrogenated carbons, 16 to methyl carbons, 1 methylene carbon and 1 methyl carbon.163,1; 101.4; 166.3 and 94.9 were assigned to the C-5 aromatic carbons; C-6; C-7 and C-8, respectively, suggesting to treat the A-ring substitution pattern of oxygenated flavones in C-5 and C-7. The signal at δc 132.1 was corresponding to the C-2 'and C-6' carbons and the signal at δc 116.70 was attributed to the H-3 'and H-5' hydrogens; being these characteristic of ring B of flavones. The signals at δc 158.7; 136.4 and 179.6 were assigned to the C-2 carbons; C-3 and C-4 respectively, being compatible with an oxygenated flavone at C-3. According to the ¹³C data, it was also possible to suggest the presence of two osydic units, being a glucose and a rhamnose, this was due to the signs of the carbonos anomeric in δc 103,6 and δc 102,4 and also to the carbon methylenic in 68,6 and carbon in δc17.8, and, in addition to the set of signals between δc 68.6 and δc 78.2, suggested the presence of rutinose.
In the ¹H spectrum, two doublets δ H 6.38 and δ H 6.20 (J = 2.0 Hz), characteristic of hydrogen H-8 and H-6 of hydrogen peroxide flavones at positions 5 (J = 8.8 Hz) which was assigned to the hydrogens H-2 'and H-6', a doublet in δ H 6.93 (J = 8.8 Hz) which was assigned to the H-3 and H-5 hydrogens of the B-ring of flavones in an AA'BB' system. In addition, a doublet in δ H 5.37 (J = 1.6 Hz) was observed in the spectrum and one in δ H 4, 57, corresponding to the hydrogen anomeric da glicose and rhamnose respectively. The presence of a doublet in δ H 4.21 (dd, J = 3.6, 1.6 Hz) and another in δ H 1.10 (d, J = 6.0 Hz) were attributed to methylene and methyl hydrogels respectively. These data corroborate with an osteoid unit glucose-linked aramenosis. The coupling constant J = 1.6 Hz was consistent with an α-rutinoside unit.
The correlations observed in the Er-1 HMBC contour map of the hydrogen in δ H 5.37 (H-1 ") with the C-3 carbon in δc 136.4 at three bonds confirmed the insertion of the osydica rutinosidase in C-3. After these analyzes, it was possible to conclude that Er-1 is the camphorol-3-O-rutinoside.
The substance encoded as Er-2 was isolated as a brown solid with 15 mg. In the 13 C-APT spectrum obtained at 100 MHz in CD 3 OD of Er-2 was shown to be quite similar to Er-1 differing only in the chemical shifts at δc 117.8; δc 145.9 and δc 149.9 which were assigned to the C-2 'aromatic carbons; C-3 'and C-4' respectively, when compared to these same carbons in Er-1, it was possible to suggest the insertion of a hydroxyl in C-3 'due to an ortho effect of electron donor group that promoted protection in C -2 'and C-4'.
In the ¹H spectrum, it was shown to be quite similar to Er-1, differentiating only in the absence of the signal in δ H 6.93 (d, J = 8.8 Hz), where a C-3 'group was suggested. It was also observed that the chemical shifts and coupling constants values were δ H 7.67 (d, J = 2.0 Hz), δ H 6.89 (d, J = 8.4 Hz) and δ H 7.63 (dd, J = 8,4; 2,0 Hz), which were attributed to the hydrogen H-2 ', H-5' and H-6 ', respectively, being suggestive of the presence of an ABX system in ring B of flavones. After these analyzes, it was possible to conclude that Er-2 is quercetin-3-O-β-Dglucopyranoside-α-L-raminoside.