**2.4. Betulinic acid quantification**

GC analysis was performed on 6890N (Agilents Technologies, Network series) equipped whit a HP-5 column (30 x 0,25mm; 0,25µm liquid phase). Oven temperature program of 70o C - 300o C at 5o C/min; carrier gas: helium 11,3L/min; split mode of (20:1) and finally held for 30min. The mass spectrometer unit was performed with the same conditions the GC analysis. The calibration curve of the GC / FID was made in triplicate from different concentrations of esterified betulinic acid standard (0.1 to 1µg.mL-1) and the curve was constructed using the average values of the detector response. The detector response was linear to the concentration internal of 0.1 to 1µg.mL-1 (r2 = 0.999, Figure 2a).

HPLC grade acetonitrile was purchased from TEDIA (Brazil); 0,05% TFA (Trifluoroacetic acid) fromVetec (Brazil).WaterwaspurifiedbyMilli-Qplus systemfromMilipore (Milford,MA,USA). BetulinicacidwaspurchasedfromCarlRoth(Karlsruhe,Germanywith99%).The30mgethanol extracts were then dissolved in 5mL of mobile phase. The mobile phase consisted of a gradient of 0,05%aqueous trifluoroacetic acid: acetonitriledeliveredat a 1.0mL.min-1 as follows initial(t= 0 min) 30:70, linear gradient over 20min to 15:85, linear gradient over 10min to 100:0 and a new linear gradient over 20min (30:70); 40min as total time of analysis. Flow rate was 1mL.min-1. Quantificationwasperformedusingthedetector setatawavelengthof210nm.Injectionvolume was 30µl. The peak of betulinic acid was identified in each chromatogram from of the ethanol extracts monthly (twelve months) with the help of injection of the standard solution of betulin‐ ic acid or comparison of the UV spectrum. The calibration curve of the HPLV-UV was made in triplicate fromdifferent concentrationsofbetulinic acidstandard(0.1 to1µg.mL-1) andthe curve was constructed using the average values of the detector response. The detector response was linear to the concentration internal of 0.1 to 0,5µg.mL-1 (r2 = 0.9994, Figure 2b).

CH2N2 with 100% yield. Those samples were dissolved in CH2Cl2 in concentrations 1.4mg/mL and injected for GC-FID and GC-MS analysis. The identification of methylated betulinic acid in extracts was done with use Wiley and NBS peak matching library search system. Authentic standard of the betulinic acid and data reported in the literature were also used for further

Healthy leaves of *Eugenia florida* and adults were collected during 12 months [August 2009 to July 2010] on the campus Oswaldo Cruz Foundation, state of Rio de Janeiro. The specie identification was carried out by biologist Sergio Monteiro of Oswaldo Cruz Foudantion [Laboratory of Production and Processing of Raw Plant (LPBMPV)], and a voucher specimen was deposited in the Herbarium of the Botanical Garden of Rio de Janeiro with the number

A solution of diazomethane (CH2N2) in ether was prepared and added (excess) in drops of the solutions of extracts, EF-1 and standard (1mg) CHCl3 or MeOH. The resulting solutions were allowed to stand for 12 hours and the ether removed by passing a stream of N2 [Leonard, Lygo,

GC analysis was performed on 6890N (Agilents Technologies, Network series) equipped whit a HP-5 column (30 x 0,25mm; 0,25µm liquid phase). Oven temperature program of 70o

The mass spectrometer unit was performed with the same conditions the GC analysis. The calibration curve of the GC / FID was made in triplicate from different concentrations of esterified betulinic acid standard (0.1 to 1µg.mL-1) and the curve was constructed using the average values of the detector response. The detector response was linear to the concentration

HPLC grade acetonitrile was purchased from TEDIA (Brazil); 0,05% TFA (Trifluoroacetic acid) fromVetec (Brazil).WaterwaspurifiedbyMilli-Qplus systemfromMilipore (Milford,MA,USA). BetulinicacidwaspurchasedfromCarlRoth(Karlsruhe,Germanywith99%).The30mgethanol extracts were then dissolved in 5mL of mobile phase. The mobile phase consisted of a gradient of 0,05%aqueous trifluoroacetic acid: acetonitriledeliveredat a 1.0mL.min-1 as follows initial(t= 0 min) 30:70, linear gradient over 20min to 15:85, linear gradient over 10min to 100:0 and a new linear gradient over 20min (30:70); 40min as total time of analysis. Flow rate was 1mL.min-1. Quantificationwasperformedusingthedetector setatawavelengthof210nm.Injectionvolume was 30µl. The peak of betulinic acid was identified in each chromatogram from of the ethanol extracts monthly (twelve months) with the help of injection of the standard solution of betulin‐ ic acid or comparison of the UV spectrum. The calibration curve of the HPLV-UV was made in triplicate fromdifferent concentrationsofbetulinic acidstandard(0.1 to1µg.mL-1) andthe curve

= 0.999, Figure 2a).

C/min; carrier gas: helium 11,3L/min; split mode of (20:1) and finally held for 30min.

C -

identification as described.

**2.2. Plant material**

186 Column Chromatography

RB 328061.

**2.3. Methylation**

Procter, 1995].

C at 5o

300o

**2.4. Betulinic acid quantification**

internal of 0.1 to 1µg.mL-1 (r2

**Figure 2.** Calibration curve of Betulinic acid, A: GC-FID and B: HPLC-UV
