**2. Materials and methods**

#### **2.1 Reagents and solvents**

190 Chromatography and Its Applications

(**3**), a structural isomer of **2** and a derivative, 15-deacetylsergeolide (**4**) (Polonsky *et al*. 1984), from the leaves. Since confirmation of the structure of **2** by x-ray crystallography (Schpector *et al*. 1994) and the systematic application of two-dimensional NMR techniques to the identification of components of *P. sprucei* (Vieira *et al*. 2000, Andrade-Neto *et al*. 2007), neither sergeolide nor its derivative have ever again been described and may be erroneous

Chemically, quassinoids are degraded triterpene compounds which are frequently highly oxygenated. Many quassinoids exhibit a wide range of biological activities *in vitro* and/or *in vivo,* including antitumor, antimalarial, antiviral, anti-inflammatory, antifeedant, insecticidal, amoebicidal, antiulcer and herbicidal activities. For instance, bruceantin (**5**), brusatol (**6**), simalikalactone D (**7**), quassin (**8**) and glaucarubinone (**9**) are some of the most well-studied quassinoids and exhibit a wide range of biological activities (Guo *et al*. 2005).

**O O**

**R= COC(CH3)OHCH2CH3**

**OR**

**O O**

**R= COCH(CH3)C2H5**

**OR**

**O**

**OH**

**HO**

**OH**

**7:** 

**O**

**HO**

**9:**

**O**

Fig. 2. Quassinoids bruceantin, simalikalactone D, quassin, brusatol and glaucarubinone.

antileukemic, antifeedant and leishmanicidal (Moretti *et al*. 1982; Nunomura, 2006).

Isobrucein B (Fandeur et al. 1985) and neosergeolide (Andrade-Neto *et al*. 2007) display significant *in vitro* antimalarial activity to the human malaria parasite *P. falciparum.* Recently, the *in vitro* antimalarial activities of isobrucein B and neosergeolide were shown to be comparable to antimalarial drugs quinine and artemisinin (Silva *et al*. 2009). According to this same *in vitro* study, isobrucein B and neosergelide are as cytotoxic or as much as an order of magnitude more cytotoxic than the antitumor drug doxorubicine towards several human tumor strains. Additionally, isobrucein B has been shown to have important

Bertani *et al*. (2005) conveyed concern about the toxicity of infusions and other preparations based on different parts of *P. sprucei* which is recognized in Amazonian traditional medicine in general. Additionally, these authors were critical of the absence of knowledge of the toxicity of infusions prepared from this species and lack of information available on the

**OH O**

**OH**

**O O**

**6: R= COCH=C(CH3)2**

**R= COCH=CCH(CH3)2 CH3**

> **O O H**

**OMe**

**H**

**8** 

**O O**

**5:** 

**CO2CH3 OR**

**O**

**OH**

**HO**

structures.

**O**

**HO**

**MeO**

Acetonitrile, HPLC grade, was purchased from Mallinckrodt Baker, Inc. (Xalostoc, Mexico). The water used in all experiments was purified on a Milli-Q Plus System (Millipore, Bedford, MA, USA).

#### **2.2 Isolation of isobrucein B (1) and neosergeolide (2)**

Two collections were performed on the main campus of the University of Amazonas, in Manaus, Amazonas State, Brazil, in January and July of 1999. Voucher specimens are deposited at the UFAM Herbarium (Silva 5729 & 5730) and INPA Herbarium (223883). Identification was performed by Dr. Wayt Thomas as *Picrolemma sprucei* Hook. f. (Wayt Thomas, personal communication). Roots and stems were cut into small pieces while fresh and allowed to dry in the shade and were then ground. Air-dried powdered stems (890 g) were extracted 3 times by maceration in hexanes at room temperature (1 week per extraction). After concentration, hexane extract (4.79 g) was obtained. Next, the stems were repeatedly infused in boiling water (Polonsky 1982) which resulted in aqueous solution (20 L). The aqueous solution was concentrated (2.0 L) then continuously extracted with chloroform (40 h), that after total evaporation yielded chloroform extract (10.8 g). Chloroform extract was purified on a column of silica gel which was eluted first with chloroform (100 %), then a gradient of chloroform/methanol 99:1–70:30 (600 mL), 70:30– 50:50 (600 mL), 50:50–25:75 (600 mL), and 25:75–100 % methanol (600 mL) and resulted in 171 collected fractions that were combined based on thin-layer chromatography (TLC) analysis to yield 11 fractions. Fraction 9 (1.87 g) was purified on a column of silica gel which was eluted first with 100 % hexane, then 80:20 hexane/chloroform (180 mL), 15:80:5 hexane/chloroform/acetone (800 mL), 10:80:10 hexane/chloroform/acetone (400 mL), 10:70:20 hexane/chloroform/acetone (1440 mL), 10:60:30 hexane/chloroform/acetone (500 mL), 10:50:40 hexane/chloroform/acetone (720 mL), acetone (500 mL), and methanol (500 mL) which resulted in 69 fractions that were combined based on TLC analysis. Combined fraction 42-50 (360 mg) was re-crystallized from methanol/water and yielded colorless crystals which were identified as pure **2** (73.9 mg) based on their spectral properties. The supernatant was re-dissolved in methanol and the insoluble material was washed and filtered resulting in **1** (62.0 mg), a white solid, which was identified based on its spectral properties. The isolation of **1** and **2** yielded 0.57% and 0.68 %, respectively. The compounds **1** and **2** were identified on the basis of their IV, MS and NMR (1H, 13C, HOMOCOSY, HMQC, HMBC and NOESY experiments) spectra analysis.

Quantification of Antimalarial Quassinoids Neosergeolide and Isobrucein B

in Stem and Root Infusions of *Picrolemma sprucei* Hook F. by HPLC-Uvanalysis 193

Fig. 3. A: chromatograms of root infusions with (back trace) and without (front trace)

(tR= 14.0 min) at 254 nm. C=chromatogram of neosergeolide (tR= 25.3 min) at 254 nm.

standard sample concentrations Y and X, respectively (figure 3) at 254 nm.

addition of neosergeolide and isobrucein B at 254 nm. B: chromatogram of pure isobrucein B

Several injections of standard solution were performed and then average areas were calculated for each individual concentration injected for isobrucein B (1) and neosergeolide (2). The calibration curves in the determination of **1** and **2** in *P. sprucei* stem and root infusions (Figure 4A and 4B, respectively) used in the determination of these components in *P. sprucei* stem and root infusions were obtained by linear regression performed on the average areas versus

#### **2.3 Preparation of root and stem infusions**

*P. sprucei* infusions were prepared based on a popular recipe which is used to provoke spontaneous abortions and with which toxic effects are associated according to locals. Stems are the part most commonly used in these remedies. Shade-dried, ground root or stem (9.0 g) was placed in a beaker and boiling deionized water (1.0 mL) was added. The beaker was covered and allowed to stand for 10 min. After this time, the contents of the beaker was filtered hot in a funnel with filter paper which resulted in root and stem infusions. A single infusion was prepared from powdered, dried roots and another from powdered stems obtained from mature plants.

### **2.4 Calculation of extractives**

Infusion as prepared above was totally evaporated using rotary evaporation under vacuum and a heated bath (< 50 ºC), then freeze-drying. The resulting dry extract was weighed and divided by the mass of plant material used (9.0 g) in the preparation of each infusion and expressed as a percentage (w/w) of extractives.

### **2.5 Preparation of samples of infusions for HPLC analysis**

Freeze-dried extracts were dissolved in water to yield final concentrations of stem and root extracts of 445 and 911 mg.L-1, respectively.

#### **2.6 Preparation of standard solutions of isobrucein B (1) and neosergeolide (2)**

Stock solutions of **1** and **2** were prepared at 0.63 g.L-1 and 0.50 g.L-1, respectively, in methanol. Calibration standards were obtained by appropriate dilution of the stock solutions with methanol. For **1**, the concentrations used in calibration were 100, 50, 25, 10 and 5.0 mg.L-1. For **2**, the concentrations used in calibration were 20, 10, 5.0 and 2.5 g.L-1. All standard solutions were stored at -20 °C until analysis and protected from light, remaining stable for at least three months.

#### **2.7 Apparatus and chromatographic conditions**

The liquid chromatography system consisted of an LC-10 Shimadzu, with a SPD-10A UV detector, LC-10AVp quaternary pump, SIL- 10A autosampler and a CBM-10A system controller (Kyoto, Japan). A Supelcosil LC-18 analytical column (250 mm × 4.6 mm i.d., 5 μm particle size) from Supelco (Bellefonte, PA, USA) was used for separation of **1** and **2.** The mobile phase consisted of a gradient of acetonitrile:0.05 % aqueous trifluoroacetic acid delivered at 1.0 mL.min-1 as follows: initial (ti=0 min) 10:90, then linear gradient over 20 min to 25:75, and this composition was maintained (isocratic) until the end of each run (tf=30 min). Flow rate was 1 mL.min-1. Quantification was performed using the detector set at a wavelength of 254 nm. Injection volume was 50 μL.

#### **2.8 Analysis of Infusions by HPLC-UV and calibration curve**

Chromatograms of pure **1** and **2** presented retention times of approximately 14 and 25 min, respectively. The peaks corresponding to **1** and **2** were identified in each chromatogram of the infusions with the help of injection of the standard solutions of **1** and **2** or with coelution (figure 3).

*P. sprucei* infusions were prepared based on a popular recipe which is used to provoke spontaneous abortions and with which toxic effects are associated according to locals. Stems are the part most commonly used in these remedies. Shade-dried, ground root or stem (9.0 g) was placed in a beaker and boiling deionized water (1.0 mL) was added. The beaker was covered and allowed to stand for 10 min. After this time, the contents of the beaker was filtered hot in a funnel with filter paper which resulted in root and stem infusions. A single infusion was prepared from powdered, dried roots and another from powdered stems

Infusion as prepared above was totally evaporated using rotary evaporation under vacuum and a heated bath (< 50 ºC), then freeze-drying. The resulting dry extract was weighed and divided by the mass of plant material used (9.0 g) in the preparation of each infusion and

Freeze-dried extracts were dissolved in water to yield final concentrations of stem and root

Stock solutions of **1** and **2** were prepared at 0.63 g.L-1 and 0.50 g.L-1, respectively, in methanol. Calibration standards were obtained by appropriate dilution of the stock solutions with methanol. For **1**, the concentrations used in calibration were 100, 50, 25, 10 and 5.0 mg.L-1. For **2**, the concentrations used in calibration were 20, 10, 5.0 and 2.5 g.L-1. All standard solutions were stored at -20 °C until analysis and protected from light, remaining

The liquid chromatography system consisted of an LC-10 Shimadzu, with a SPD-10A UV detector, LC-10AVp quaternary pump, SIL- 10A autosampler and a CBM-10A system controller (Kyoto, Japan). A Supelcosil LC-18 analytical column (250 mm × 4.6 mm i.d., 5 μm particle size) from Supelco (Bellefonte, PA, USA) was used for separation of **1** and **2.** The mobile phase consisted of a gradient of acetonitrile:0.05 % aqueous trifluoroacetic acid delivered at 1.0 mL.min-1 as follows: initial (ti=0 min) 10:90, then linear gradient over 20 min to 25:75, and this composition was maintained (isocratic) until the end of each run (tf=30 min). Flow rate was 1 mL.min-1. Quantification was performed using the detector set at a

Chromatograms of pure **1** and **2** presented retention times of approximately 14 and 25 min, respectively. The peaks corresponding to **1** and **2** were identified in each chromatogram of the infusions with the help of injection of the standard solutions of **1** and **2** or with co-

**2.6 Preparation of standard solutions of isobrucein B (1) and neosergeolide (2)** 

**2.3 Preparation of root and stem infusions** 

expressed as a percentage (w/w) of extractives.

extracts of 445 and 911 mg.L-1, respectively.

**2.7 Apparatus and chromatographic conditions** 

wavelength of 254 nm. Injection volume was 50 μL.

**2.8 Analysis of Infusions by HPLC-UV and calibration curve** 

**2.5 Preparation of samples of infusions for HPLC analysis** 

obtained from mature plants.

**2.4 Calculation of extractives** 

stable for at least three months.

elution (figure 3).

Fig. 3. A: chromatograms of root infusions with (back trace) and without (front trace) addition of neosergeolide and isobrucein B at 254 nm. B: chromatogram of pure isobrucein B (tR= 14.0 min) at 254 nm. C=chromatogram of neosergeolide (tR= 25.3 min) at 254 nm.

Several injections of standard solution were performed and then average areas were calculated for each individual concentration injected for isobrucein B (1) and neosergeolide (2). The calibration curves in the determination of **1** and **2** in *P. sprucei* stem and root infusions (Figure 4A and 4B, respectively) used in the determination of these components in *P. sprucei* stem and root infusions were obtained by linear regression performed on the average areas versus standard sample concentrations Y and X, respectively (figure 3) at 254 nm.

Quantification of Antimalarial Quassinoids Neosergeolide and Isobrucein B

6 28.5 1.86 (ddd; 14.7; 12.1; 2.6); 2.40 (ddd;

**1** and **2** are presented in tables 1 and 2 respectively.

**3. Results and discussion** 

1Moretti et al. (1982).

isobrucein B (**1**).

in Stem and Root Infusions of *Picrolemma sprucei* Hook F. by HPLC-Uvanalysis 195

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

**Carbon (C) (H) δ Literature (CDCl3/Py-5%)1**

1 81.1 4.17 (s) 81.3 4.26 2 197.0 - 197.6 - 3 124.3 6.11 (q; 2.8; 1.0) 124.5 6.11 4 163.0 - 162.6 - 5 51.6 2.92 (d; 12.1) 43.4 2.91

7 83.1 4.75 (d) 81.7 4.75 8 45.5 - 45.8 - 9 42.8 2.34 (d; 4.0) 42.4 2.38 10 47.5 - 47.7 - 11 72.4 4.75 (sl) 74.3 4.75 12 75.8 4.28 (s) 75.1 4.12 13 80.5 - 81.7 - 14 43.5 3.04 (d; 12.4) 52.3 3.03 15 66.6 6.31 (sl) 67.8 6.30 16 167.0 - 167.6 - 29 22.5 1.96 (d; 1.0) 22.4 1.95 19 11.6 1.18 (s) 11.3 1.18 30 73.3 3.75 (dd; 7.7; 2.0); 4.81 (d; 7.7) 73.0 3.75; 4.81 21 172.6 - 169.5 - 1' 169.0 - 170.7 - 2' 20.4 2.09 (s) 20.5 2.08 OMe (5') 20.4 3.84 (s) - 3.83 H-(OH-1) - 4.54 (s) - - H-(OH-12) - 3.25 (s) - -

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

**C H** 

14.7; 2.8; 2.8) 28.2 1.86; 2.41

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 calibration of each quassinoid.

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