**3.1. Extraction of betulinic acid**

The leaves of *Eugenia florida* (17.1 kg) were dried at 400C, ground and subjected to soxhlet extraction with ethanol. The diluted extract was removed under reduced pressure (4.7 g). An aliquot of the methanol extract (200 mg) was dissolved in methanol (20ml) and recrystallized using mixtures of CHCl3 and MeOH. Recrystallization was obtained a white crystal (EF – 1; 50mg).

corresponding to betulinic acid was calculated. From these average areas, percentage compo‐ sition of the betulinic acid in the extract were calculated using the linear equation generated during calibration of betulinic acid (Figure 2) carried out in HPLC-UV and GC-FID (Table 1).

Analysis of the Presence of the Betulinic Acid in the Leaves of *Eugenia florida* by…

http://dx.doi.org/10.5772/55868

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**Table 1.** Quantification (w/w) of betulinic acid present in ethanol extracts from leaves *of Eugenia florida* determined

Several activities are being attributed to betulinic acid, however, despite all of their potential pharmacological, it is still obtained by extraction of the bark and heartwood of some [Soler, 1996], synthetic processes [Evers et al., 1996] and by biotransformation [Galgon, 2005]. Unlike these traditional species whose income was less than 3%, we found that betulinic acid was present in all extracts analyzed (table 1), with yields well above those found in the literature.

The betulinic acid level in the *E. florida* leaves increased significantly in the May, June, Jully (autumn - winter) and, September, October and November (winter) which was mainly due to the accumulation of this compound in vegetal tissue. Some authors related with the pentacyclic triterpenes, just as betulinic, acid ursolic, acid, β-amyrine and lupeol, are supposed to be toxic to insects, due to their ability to inhibit acyl chain packing in the lipid bilayers of the insect

These fluctuations observed in the months described in Table 1 may be related to the chemical ecology of *Eugenia florida* as, for example, the attraction of pollinators or the reproductive

membranes [Rodriguez *et al*., 1997; Prades et al., 2011].

**Month (year) CG-FID (%) HPLC (%)** August (2009) 7.43 8.01 September (2009) 16.83 8.57 October (2009) 26.27 11.18 November (2009) 17.97 8.36 December (2009) 8.24 4.83 January (2010) 6.90 2.79 February (2010) 7.92 5.12 March (2010) 23.03 6.12 April (2010) 9.68 6.31 May (2010) 14.85 9.16 June (2010) 19.33 9.99 Jully (2010) 15.23 5.62

by GC-FID and HPLC-UV at 210nm

phenology of the specimens

**4. Conclusions**

EF-1 was analyzed by spectrophotometer 1H and 13C NMR (Bruker AC 200, 200MHz) using as solvent chloroform (CDCl3) and methanol (CD3OD) deuterated at a ratio of 9:1 to tetrame‐ thylsilane (TMS) as internal reference standard. An aliquot of EF-1 (5 mg) was methylated with diazomethane and subjected to mass spectrometry (MS; Agilent Technologies). The spectral data obtained were compared with the literature [Oliveira *et al*., 2006].

The substance showed an EF-1 in the form of white crystals and the IR spectrum showed a broad band at 3450cm-1 by a characteristic of hydroxyl groups and acid, a broad band at 2942cm-1 one of alkyl groups and bands at 1686cm-1 and 1639cm-1 corresponding respectively to the axial deformation of carbonyl acid and alkene.

The information that led to elucidation of the structure was obtained from experiments nuclear magnetic resonance spectra [DEPT, HMQC, 1 H-1 H COSY (homonuclear correlation spectro‐ scopy) and HMBC experiment] which indicate a known pattern of the terpenes series lupanos (Nick *et al*., 1994, Mahato, Kundu 1994; Budzikiewicz *et al.*, 1964). The 1 H NMR spectrum showed two signals of multiplet in δH 4.69 and 4.58, referring to vinyl hydrogen (H-20), δH1.66 a signal corresponding to the methyl group bonded to carbon and fifth signals sp2 corre‐ sponding to the methyl tertiary (δH 0.74; 0.85, 0.94, 0.96 and 1.00). The 13 C NMR spectrum confirmed the presence of signals in vinyl 152.02 and 110.15 ppm (double bond), carbonyl acid in 180.03ppm and secondary alcohol in 79.69 ppm [Nick *et al*., 1994; Mahato, Kundu, 1994].

The methylation EF-1 with diazomethane promoted the removal of hydrogen from the carboxyl acid and incorporation of a methyl group from the diazomethane, leading to formation of an ester, molecular weight 470. The derivatization and the formation of the ester are ideal possible to decrease the molecular interactions between the sample and a chromato‐ graphic column and thus decrease the retention time. An aliquot of esterified EF-1 (1.4mg) was subjected to MS electron impact (70 eV). The MS spectrum of esterified EF-1 confirmed the presence of a terpene class of lupanos due to the absence of peaks m/z 218 and m/z 203 characteristic of the series oleanane and ursane (rearrangement retro Diels-Alder ring C). The presence of the methyl ester group at C-28 is confirmed by the ion m/z 262 (10%). Other peaks were obtained m/z 208 (5%), m/z 190 (10%) and m/z 189 (100%) from the break ring C and the molecular ion m/z 470 5% [Budzikiewicz *et al*., 1964]. The spectral data obtained from the EF-1 and the ester data were similar to those observed in the literature to betulinic acid [Nick *et al*., 1994, Mahato, Kundie 1994; Budzikiewicz *et al.*, 1964].

After calibration with standard of betulinic acid, the monthly extracts from leaves of *Eugenia florida* were analyzed. Those extracts were analyzed in triplicate and the average areas corresponding to betulinic acid was calculated. From these average areas, percentage compo‐ sition of the betulinic acid in the extract were calculated using the linear equation generated during calibration of betulinic acid (Figure 2) carried out in HPLC-UV and GC-FID (Table 1).


**Table 1.** Quantification (w/w) of betulinic acid present in ethanol extracts from leaves *of Eugenia florida* determined by GC-FID and HPLC-UV at 210nm
