**4. Conclusions**

**3. Results and discussion**

188 Column Chromatography

**3.1. Extraction of betulinic acid**

crystal (EF – 1; 50mg).

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

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

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

The information that led to elucidation of the structure was obtained from experiments nuclear

scopy) and HMBC experiment] which indicate a known pattern of the terpenes series lupanos

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‐

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*.,

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

H-1

H COSY (homonuclear correlation spectro‐

H NMR spectrum

C NMR spectrum

data obtained were compared with the literature [Oliveira *et al*., 2006].

(Nick *et al*., 1994, Mahato, Kundu 1994; Budzikiewicz *et al.*, 1964). The 1

sponding to the methyl tertiary (δH 0.74; 0.85, 0.94, 0.96 and 1.00). The 13

to the axial deformation of carbonyl acid and alkene.

1994, Mahato, Kundie 1994; Budzikiewicz *et al.*, 1964].

magnetic resonance spectra [DEPT, HMQC, 1

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

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 phenology of the specimens

It is possible that the increased concentration of betulinic acid in the month of March is due to the large amount of rainfall characteristic of the Rio de Janeiro, state. However, more research is needed to determine whether other factors may be influencing the concentration of this metabolite, verify that specimens from other regions have the same or different behavior and examine whether the effect of the solvent can affect the increase in the concentration of this metabolite

[3] Consolini, A. E, Baldini, O. A. N, & Amat, A. G. (1999). Pharmacological basis for the empirical use of *Eugenia uniflora L.* (Myrtaceae) as anthypertensive. Journal of Ethno‐

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

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

191

[4] Evers, M, Poujade, C, Soler, F, Ribeil, Y, James, C, Lelièvre, Y, Geguen, J. C, Reisdorf, D, Morize, I, Pauwels, E, De Clercq, E, Hènin, Y, Bousseau, A, & Mayaux, J. F. Le Pecq, J.B.; Dereu N. ((1996). *Betulinic Acid Derivatives: A new class of HIV type 1 specific inhibitors with a new mode of action*. Journal of Medicinal of Chemistry , 39, 1056-68.

[5] Galgon, T, Wohlrab, W, & Drager, B. (2005). Betulinic acid induces apoptosis in skin cancer cells and differentiation in normal human keratinocytes. Experimental Der‐

[6] Galgon, T, Höke, D, & Dräger, B. (1999). *Identification and Quantification of Betulinic*

[7] Gobbo-neto, L, & Lopes, N. P. (2007). Medicinal plants: factors of influence on the content of secondary metabolites. Química Nova. Mar./Apr. São Paulo., 30(2)

[8] Hendriks, H, Wildeboer, Y. A, Engels, G, Bos, R, & Woerdenbag, H. J. (1997). The content of parthenolide and its yield per plant during the growth of *Tanacetum par‐*

[9] Junges, M. J, Fernandes, J. B, Vieira, P. C, Silva, M. F. G, & Filho, E. R. (1999). *The use*

[10] Leonard, J, Lygo, B, & Procter, G. Advanced Pratical Organic Chemistry, 2 nd ed.

[11] Lunardi, I, Peixoto, J. L. B, Silva, C. C, & Shuquel, I. T. A. Basso E.A.E; Vidotti G.J ((2001). Triterpenic Acids from Eugenia moraviana. Journal Brazilian Chemical Soci‐

[12] Mahato, S. B, & Kundu, A. P. (1994). C NMR spectra of pentacyclic triterpenoids- A

[13] Mayaux, J. F, Bousseaux, A, Panwels, R, De Clerq, E, & Pecq, J. B. (1994). *Triterpenes derivatives that block the entry of human immunodeficiency virus type I into cells.* Proceed‐

[14] Mitscher, L. A, Pillai, S, & Shankel, D. M. (2000). Some transpacific thoughts on the regulatory need for standardization of herbal medical products. Journal Food and

[15] Navarro, F. N, Souza, M. M, Neto, R. A, Golin, V, Niero, R, & Yunes, R. A. Delle Monache, F.; Cechinel Filho, Phytochemical analysis and analgesic properties of Cur‐

cuma Zedoaria grwn in Brasil. Phytomedicine, n.9, , 2002, 427-432.

compilation and some salient features. Phytochemistry , 37, 1517-1575.

*H-NMR in the structure elucidation of a new nor-lupane triterpene*. Journal Bra‐

pharmacology, , 66, 33-39.

matology. October, , 14(10), 736-743.

*Acid. Phytochem* Anal. 10. , 187-190.

*thenium.* Planta Medica , 63, 356-359.

zilian Chemical Society , 10(4), 317-320.

ings National Academy Sciences, , 91, 3564-3568.

Chapman & Hall, (1995).

Drug Anal, n. 4, , 8, 229-234.

ety, , 12, 180-183.

*of 13C and 1*
