Fig. 1. Estructure of amyrins NMR methods have indisputably become the single most important spectroscopic techniques for the identification and structure elucidation of amyrins. Several ID and 2D NMR methods are now commonly used for the characterization of pentaclyclic triterpenes. These methods incluye 1 H and 13 C-NMR, APT, DEPT, COSY, HMQC, HMBC and TOCSY. The 1 H and 13 C-NMR assignments of --amyrin are presented in Table 1, (Dias et al., 2011). 2.1.2 Detection Amyrins are found in various plants and plant materials such as leaves, bark, wood, and resins. This material has to be pre-treated prior to isolation of the target compounds. First, the plant material is usually dried, then ground into a power and sieved. Second, extractions are carried out with dichloromethane, chloroformo, n-hexane, and methanol. The samples can be subjected to alkaline hydrolysis, derivatization and separation by thin layer chromatography, and the resulting material can be directly subjected to analysis. Gas chromatography (CG) and high performance thin layer chromatography (HPTLC) techniques are the most commonly employed methods to quantitate -, -amyrin in plants. TLC provided an easy and rapid way to study plant extract profiles and partially identify compounds. The first step for the identification of -amyrin, -amyrin and 3-epi-lupeol was to compare R F values of reference standards with those of sample extracts. TLC on silica gel revealed that -amyrin on tracks 6 and 15, -amyrin on track 14 and the -, -amyrin mixture on track 16, as well as two standards, all had the same R F (Fig. 2). The -amyrin band was observed as brown, while the -amyrin band appeared violet, as did the band for the -, -amyrin mixture. TLC analysis revealed the presence of -amyrin, -amyrin and 3epi-lupeol by a comparison of the position and color of the triterpene spots with those of authentic compounds (Fig. 2). The bands of and -amyrin or their mixture were observed in all commercial resin tracks 1-5 and medicinal plant tracks 8-13, while 3-epi-lupeol track 7 was detected only in the commercial Mexican Copal resins tracks 1-4. Attempts were made to separate the -, -amyrin mixture, which had appeared homogenous on TLC, but without success. These results showed that TLC can be used as a simple method for a preliminary analysis of these triterpenes in extracts of commercial resins and plants, but cannot be employed for the analysis of the -, -amyrin mixture.

bovine serum. The BV2 cells were pretreated with test compounds for 1 h and then stimulated with 100 ng/ml LPS. After further 24 h incubation, 100 μl of the medium was used for the measurement of nitrite concentration, and the cells were used for the assessment of viability. The nitrite concentration in the medium was measured using Griess reagent (1% sulfanilamide in 5% H 3 PO 4 and 0.1% N-1-naphtyletylenediamide dihydrochloride), and the cell viability was determined with the WST assay (EZ-Cytox). WST solution was added to each 96-well plate and incubated for 2 h. The optical density (OD) was read at 450 nm. The cell viability was calculated using the following equation: % protection = 100 × (OD of LPS-sample treated cultures or OD of LPS treated cultures)/(OD of control cultures).

The separation of the diastereoisomers of the nucleoside derivatives of uridine, inosine and adenosine was performed by HPLC using chiral and no chiral columns, it was observed with the no chiral columns the resolution was good enough to determine diastereoisomeric excess. These methods were compared with H NMR, and no significant differences were observed between the three techniques. Diastereoisomeric uridine (3a), inosine (3b) and adenosine (4c) cyanohydrins were resolved byH nuclear magnetic resonance (H NMR), chiral normal phase-high-performance liquid chromatography-diode array detector (NP-HPLC-DAD) and reversed phase (RP-HPLC-DAD); these methods allowed the assesment of the percent diastereoisomeric excess (% de) of the nucleosidic cyanohydrins of 3a (4, 6 and 4), 3b (10, 8 and 6) and 4c (4, 4 and 4). To the best of our knowledge, there are no reports using analytical techniques for the separation of the epimers of 3a, 3b and 4c.