**3. Results and discussion**

194 Chromatography and Its Applications

After calibration with standard samples of isobrucein B and neosergeolide, *P. sprucei* root and stem infusions were analyzed. Samples of infusions were analyzed in triplicate and the average values of the areas corresponding to the quassinoids neosergeolide and isobrucein B were calculated. From these average areas, the concentration of each quassinoid was calculated in the root and stem infusions using the linear equation generated during


**C (mg/mL)**

y = 1.147E+07x R² = 9.993E-01

A

y = 3.193E+08x R² = 9.917E-01

B

Fig. 4. A: Calibration curve of isobrucein B (**1**); B: Calibration curve of neosergeolide (**2**).

0 0.002 0.004 0.006 0.008 0.01 0.012

**C (mg/mL)**

calibration of each quassinoid.

**Area**

0

1000000

2000000

3000000

4000000

**Area**

The quassinoids isolated from *P. sprucei* were identified by NMR techniques and compared to literature (Moretti, *et al.* 1982, Vieira, *et al.* 2000). The chemical shifts of NMR 1H and 13C of **1** and **2** are presented in tables 1 and 2 respectively.


1Moretti et al. (1982).

Table 1. Chemical shifts in NMR 1H (500 MHz, CDCl3) and NMR 13C (125 MHz, CDCl3) of isobrucein B (**1**).

Quantification of Antimalarial Quassinoids Neosergeolide and Isobrucein B

HPLC.

was isolated.

in table 3.

**4. Conclusion** 

in Stem and Root Infusions of *Picrolemma sprucei* Hook F. by HPLC-Uvanalysis 197

The authenticity of standards is a key-step in quantitative analysis, especially in plant extract analysis. In most cases, authentic standards are not available commercially and this strengths the importance of liquid chromatography. Liquid chromatography enables the isolation of authentic standards at different scales (from microgram until gram scale) and at very high purity that can be used later to perform quantitative analysis. In our study, combining open-column and planar chromatography, we were able to isolate several milligrams of each pure standard, as can be observed at figure 3, that were then used in the quantitative analysis of the quassinoids **1** and **2** in root and stem infusions of *P. sprucei* by

The structural authenticity of each standard can be confirmed by the use of modern spectroscopy techniques as MS and NMR. Although these techniques are considered complementary, normally NMR is much more informative. For instance, in our study, HMBC experiments furnished conclusive information that not sergeolide, but neosergeolide

As described in the experimental section, samples of stem and root infusions were prepared using approximately 9 g of crushed, dried stems are infused with 1 L of boiling water. HPLC analysis of *P. sprucei* stem and root infusions resulted in the concentrations presented

**Quassinoid Root infusion Stem infusion** 

isobrucein B (**1**) 32.0 67.0 14.0 29.0 neosergeolide (**2**) 0.79 1.6 0.38 0.75

Table 3. Concentrations of isobrucein B (**1**) and neosergeolide (**2**) in *P. sprucei* stem and root

Consistent with the data presented in table 1 the concentrations of both **1** and **2** are at least twice as large in root infusions as in stem infusions. Interestingly, the percentage of extractives of roots during infusion (5.1 %) is twice that of stems (2.5 %) which would seem

Comparison of root and stem infusions shows that **1** is about 40 times as concentrated as **2** in both stem and root teas on a molar basis. These data suggest that the more relevant active

The HPLC analysis of infusions (aqueous extracts) of stems and roots of *P. sprucei* revealed higher quantities of isobrucein B than neosergeolide, 40 fold, for both infusions. Considering this information and *in vitro* activity of both compounds, it is very likely that isobrucein B

to be related to the greater concentration of these constituents in the root infusion.

plays more important role for the antimalarial activity than neosergeolide.

infusions determined by HPLC-UV at 254 nm.

principle in stem and root infusions analyzed is **1**.

mg.L-1 M mg.L-1 M


1 Vieira, et al. (2000)

Table 2. Chemical shifts in NMR 1H (200 MHz, CDCl3) and NMR 13C (50 MHz, CDCl3) of neosergeolide (**2**).

The authenticity of standards is a key-step in quantitative analysis, especially in plant extract analysis. In most cases, authentic standards are not available commercially and this strengths the importance of liquid chromatography. Liquid chromatography enables the isolation of authentic standards at different scales (from microgram until gram scale) and at very high purity that can be used later to perform quantitative analysis. In our study, combining open-column and planar chromatography, we were able to isolate several milligrams of each pure standard, as can be observed at figure 3, that were then used in the quantitative analysis of the quassinoids **1** and **2** in root and stem infusions of *P. sprucei* by HPLC.

The structural authenticity of each standard can be confirmed by the use of modern spectroscopy techniques as MS and NMR. Although these techniques are considered complementary, normally NMR is much more informative. For instance, in our study, HMBC experiments furnished conclusive information that not sergeolide, but neosergeolide was isolated.

As described in the experimental section, samples of stem and root infusions were prepared using approximately 9 g of crushed, dried stems are infused with 1 L of boiling water. HPLC analysis of *P. sprucei* stem and root infusions resulted in the concentrations presented in table 3.


Table 3. Concentrations of isobrucein B (**1**) and neosergeolide (**2**) in *P. sprucei* stem and root infusions determined by HPLC-UV at 254 nm.

Consistent with the data presented in table 1 the concentrations of both **1** and **2** are at least twice as large in root infusions as in stem infusions. Interestingly, the percentage of extractives of roots during infusion (5.1 %) is twice that of stems (2.5 %) which would seem to be related to the greater concentration of these constituents in the root infusion.

Comparison of root and stem infusions shows that **1** is about 40 times as concentrated as **2** in both stem and root teas on a molar basis. These data suggest that the more relevant active principle in stem and root infusions analyzed is **1**.
