**Quantification of Antimalarial Quassinoids Neosergeolide and Isobrucein B in Stem and Root Infusions of** *Picrolemma sprucei*  **Hook F. by HPLC-UV Analysis**

Rita C. S. Nunomura1, Ellen C. C. Silva1, Sergio M. Nunomura2, Ana C. F. Amaral3, Alaíde S. Barreto3, Antonio C. Siani3 and Adrian M. Pohlit2 *1Department of Chemistry, Amazon Federal University (UFAM), Amazon, 2Coordenation of Research in Natural Products, Amazon National Institute (INPA), Amazon, 3Laboratory of Natural Products, Farmanguinhos, Oswaldo Cruz Institute Foundation (FIOCRUZ), Rio de Janeiro, Brazil* 

#### **1. Introduction**

186 Chromatography and Its Applications

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Natural products have been very important to ensure the survival of the man, since the ancient times, especially as remedies to treat different diseases. Today, despite the development of new therapies and new ways of drug development (combinatorial chemistry, ie); natural products continue to play a highly significant role in the drug discovery and development process. (Newman, Cragg, 2007).

Even though fewer drugs have been approved as therapeutical agents lately, nature still inspires the drug development for neglected diseases (malaria, tuberculosis and leishmania) and alternative therapies such as phytotherapy. In both cases, medicinal plants, plants that have been used by the folk medicine for years, are mostly studied. The World Health Organization (WHO) recognized the importance of phytotherapy and the conservation of medicinal plants that stated "the importance of conservation is recognized by WHO and its Member States and is considered to be an essential feature of national programmes on traditional medicines" (Akerele, 1991).

The successful use of some medicinal plants by local population for years, in many cases for centuries, in the treatment of diseases or symptoms associated to some diseases is the basis of the development of drugs or other therapeutical products from them. For instance, artemisinin, a very potent antimalarial, including for drug-resistant malaria strains, was isolated from *Artemisia annua* L., a plant from the traditional Chinese medicine used as remedy for chills and fever for more than 2000 years (Agtmael *et al*. , 1999).

On the other hand, there is an increasing interest for medicines from nature. This interest in products of plant origin is due to several reasons as possible side-effects from synthetic

Quantification of Antimalarial Quassinoids Neosergeolide and Isobrucein B

results indicate *P. sprucei* leaf extracts have potential as antimalarials.

**O O**

OAc **CO2CH3**

**O**

**OH**

abortions.

antihelminthic activities.

**OH**

**HO**

Fig. 1. Quassinoids from *Picrolemma sprucei* Hook.

in Stem and Root Infusions of *Picrolemma sprucei* Hook F. by HPLC-Uvanalysis 189

malaria fevers (Bertani et al. 2005, Vigneron et al. 2005, Milliken 1997), gastrointestinal problems and intestinal worms (Moretti et al. 1982, Duke & Vasquéz 1994). Also, the sale of this plant is sometimes restricted by local vendors due to its use in provoking spontaneous

Studies on the biological activity of infusions and other derivatives of *P. sprucei* have shown that extracts of this plant have important antimalarial and antihelminthic activities. Bertani *et al.* (2005) reported that a *P. sprucei* leaf infusion inhibited 78 % of *Plasmodium yoelli* rodent malaria growth *in vivo* at a dosage of 95 mg/kg. Furthermore, these same authors reported that of a total of 36 preparations from 25 traditionally used antimalarial plants from French Guiana, *P. sprucei* leaf infusion had the greatest *in vitro* activity against the human malaria parasite *Plasmodium falciparum* (median inhibition concentration, IC50=1.43 μg.mL-1). These

In 2006, Nunomura *et al*. showed that water and ethanol extracts of *P. sprucei* at concentrations of 1.3 g.L-1 were lethal (90-95 % mortality) *in vitro* towards larvae of the nematoide species *Haemonchus contortus* (Barber Pole Worm), a gastrointestinal nematode parasite found in domestic and wild ruminants. These studies lend support to popular assertions that infusions and other derivatives of *P. sprucei* have important antimalarial and

**<sup>1</sup> <sup>2</sup>**

**O**

**O**

Two quassinoids have been isolated from *P. sprucei* roots, stems and leaves and identified as isobrucein B (**1**) (Moretti et al. 1982) and neosergeolide (**2**) (Schpector *et al*.1994, Vieira *et al*. 2000). Quassinoid is the name given to any of a number of bitter substances found exclusively in the Simaroubaceae family (Polonsky 1973). Early reports on *P. sprucei*  composition from French Guiana (Moretti *et al*. 1982) described the isolation of sergeolide

**O**

**O**

**3: R = OAc 4: R = OH**

**O**

**OH HO CO2CH3**

**O O**

**R**

**O O**

OAc **CO2CH3**

**O**

**HO**

**OH**

drugs and the awareness that "natural products" are harmless. The world market for phytomedicinal products was estimated in U\$ 10 billion in 1997, with an annual growth of 6.5%. In Germany, 50% of phytomedicinal products are sold on medical prescription and the cost being refunded by health insurance. This includes pharmaceutical formulations as plant extracts or purified fractions called phytomedicines or herbal remedies. In many countries, phytomedicines or herbal remedies are controlled as synthetic drugs and they have to fulfill the same criteria of efficacy, safety and quality control (Rates, 2001).

However the quality control of phytomedicines poses a significant challenge due to the complexity of a vegetable extract and column chromatography has proved to be a very helpful and powerful technique. The quality control of *Gingko biloba* L. formulations is good example of this challenge. *Gingko* leaves contains as active compounds flavonoids and terpene lactones (gingkgolides and bilobalide) along with long-chain hydrocarbons, alicyclic acids, cyclic compounds, sterols, carotenoids, among others. Most of the quality control of *Gingko* preparations are based on column chromatography and that was reviewed elsewhere already ( Sticher, 1992; van Beek, 2002).

Column chromatography, especially high performance liquid chromatography (HPLC), has been extensively used in the quality control of plant extracts and phytomedicines formulations, because of its characteristics. The chosen technique must be able to identify the interested compounds (active principles) that are normally not volatile and, in some cases, occur at very small concentrations. Ideally this technique should also be capable to quantify the interested compounds, so one can establishes dosages for the phytomedicine formulation. The required efficiency and selectivity for qualitative and quantitative analysis of the effective components can be achieved by HPLC. Li *et al*. (2011) have recently reviewed the use of different chromatography techniques, such as HPLC, in the quality control of Chinese medicine.

Although, HPLC is a very powerful technique applied in the quality control of medicinal plants, it is necessary to properly identify the active principles of the medicinal plant. This is achieved combining the use of HPLC or other separation technique with a biological test. The search for antimalarials from medicinal plants is one of the most successful examples of this combination as mentioned earlier. In the Amazon region, there are a large number of plants popularly used against malaria or associated symptoms (fever for instance). Milliken (1997) has identified over hundreds of antimalarial plants used by local population in the Amazon region. Many of these plants remained up to now without a study that could confirm their antimalarial activity.

From the fewer plants studied so far, *Picrolemma sprucei* Hook. f., has been studied by our research group. Herein we described the use of HPLC in the quality control of the antimalarial quassinoids, neosergeolide and isobrucein B, the active principles of this species.

*Picrolemma sprucei* Hook. f. (*P. pseudocoffea* Ducke is a commonly cited pseudonym) is a widely distributed and important Amazonian medicinal plant. It is known in the Amazon region by common names which call attention to its resemblance to the coffee plant: *sachacafé* in Peru (Duke & Vasquéz 1994), *caferana* in Brazil (Silva et al. 1977) and *café lane* or *tuukamwi* in French Guiana (Grenand et al. 1987). Infusions of roots, stems, and leaves of *P. sprucei* are traditionally used in different dosages and preparations for the treatment of

drugs and the awareness that "natural products" are harmless. The world market for phytomedicinal products was estimated in U\$ 10 billion in 1997, with an annual growth of 6.5%. In Germany, 50% of phytomedicinal products are sold on medical prescription and the cost being refunded by health insurance. This includes pharmaceutical formulations as plant extracts or purified fractions called phytomedicines or herbal remedies. In many countries, phytomedicines or herbal remedies are controlled as synthetic drugs and they have to fulfill

However the quality control of phytomedicines poses a significant challenge due to the complexity of a vegetable extract and column chromatography has proved to be a very helpful and powerful technique. The quality control of *Gingko biloba* L. formulations is good example of this challenge. *Gingko* leaves contains as active compounds flavonoids and terpene lactones (gingkgolides and bilobalide) along with long-chain hydrocarbons, alicyclic acids, cyclic compounds, sterols, carotenoids, among others. Most of the quality control of *Gingko* preparations are based on column chromatography and that was reviewed elsewhere

Column chromatography, especially high performance liquid chromatography (HPLC), has been extensively used in the quality control of plant extracts and phytomedicines formulations, because of its characteristics. The chosen technique must be able to identify the interested compounds (active principles) that are normally not volatile and, in some cases, occur at very small concentrations. Ideally this technique should also be capable to quantify the interested compounds, so one can establishes dosages for the phytomedicine formulation. The required efficiency and selectivity for qualitative and quantitative analysis of the effective components can be achieved by HPLC. Li *et al*. (2011) have recently reviewed the use of different chromatography techniques, such as HPLC, in the quality control of

Although, HPLC is a very powerful technique applied in the quality control of medicinal plants, it is necessary to properly identify the active principles of the medicinal plant. This is achieved combining the use of HPLC or other separation technique with a biological test. The search for antimalarials from medicinal plants is one of the most successful examples of this combination as mentioned earlier. In the Amazon region, there are a large number of plants popularly used against malaria or associated symptoms (fever for instance). Milliken (1997) has identified over hundreds of antimalarial plants used by local population in the Amazon region. Many of these plants remained up to now without a study that could

From the fewer plants studied so far, *Picrolemma sprucei* Hook. f., has been studied by our research group. Herein we described the use of HPLC in the quality control of the antimalarial quassinoids, neosergeolide and isobrucein B, the active principles of this

*Picrolemma sprucei* Hook. f. (*P. pseudocoffea* Ducke is a commonly cited pseudonym) is a widely distributed and important Amazonian medicinal plant. It is known in the Amazon region by common names which call attention to its resemblance to the coffee plant: *sachacafé* in Peru (Duke & Vasquéz 1994), *caferana* in Brazil (Silva et al. 1977) and *café lane* or *tuukamwi* in French Guiana (Grenand et al. 1987). Infusions of roots, stems, and leaves of *P. sprucei* are traditionally used in different dosages and preparations for the treatment of

the same criteria of efficacy, safety and quality control (Rates, 2001).

already ( Sticher, 1992; van Beek, 2002).

Chinese medicine.

species.

confirm their antimalarial activity.

malaria fevers (Bertani et al. 2005, Vigneron et al. 2005, Milliken 1997), gastrointestinal problems and intestinal worms (Moretti et al. 1982, Duke & Vasquéz 1994). Also, the sale of this plant is sometimes restricted by local vendors due to its use in provoking spontaneous abortions.

Studies on the biological activity of infusions and other derivatives of *P. sprucei* have shown that extracts of this plant have important antimalarial and antihelminthic activities. Bertani *et al.* (2005) reported that a *P. sprucei* leaf infusion inhibited 78 % of *Plasmodium yoelli* rodent malaria growth *in vivo* at a dosage of 95 mg/kg. Furthermore, these same authors reported that of a total of 36 preparations from 25 traditionally used antimalarial plants from French Guiana, *P. sprucei* leaf infusion had the greatest *in vitro* activity against the human malaria parasite *Plasmodium falciparum* (median inhibition concentration, IC50=1.43 μg.mL-1). These results indicate *P. sprucei* leaf extracts have potential as antimalarials.

In 2006, Nunomura *et al*. showed that water and ethanol extracts of *P. sprucei* at concentrations of 1.3 g.L-1 were lethal (90-95 % mortality) *in vitro* towards larvae of the nematoide species *Haemonchus contortus* (Barber Pole Worm), a gastrointestinal nematode parasite found in domestic and wild ruminants. These studies lend support to popular assertions that infusions and other derivatives of *P. sprucei* have important antimalarial and antihelminthic activities.

Fig. 1. Quassinoids from *Picrolemma sprucei* Hook.

Two quassinoids have been isolated from *P. sprucei* roots, stems and leaves and identified as isobrucein B (**1**) (Moretti et al. 1982) and neosergeolide (**2**) (Schpector *et al*.1994, Vieira *et al*. 2000). Quassinoid is the name given to any of a number of bitter substances found exclusively in the Simaroubaceae family (Polonsky 1973). Early reports on *P. sprucei*  composition from French Guiana (Moretti *et al*. 1982) described the isolation of sergeolide

Quantification of Antimalarial Quassinoids Neosergeolide and Isobrucein B

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

HMQC, HMBC and NOESY experiments) spectra analysis.

detection (UV).

Bedford, MA, USA).

**2. Materials and methods 2.1 Reagents and solvents** 

in Stem and Root Infusions of *Picrolemma sprucei* Hook F. by HPLC-Uvanalysis 191

quassinoid composition of these infusions in the study on toxicity published by Fandeur *et al*. (1985), which focused only on the acute toxicity and antimalarial activity of isolated quassinoid components o *P. sprucei* and not on toxicity and antimalarial activity of infusions. Additional studies are needed to prove the *in vivo* efficacy and pharmacological activity of these infusions as antimalarials with focus on the dose-effect and dose-response to define the levels of toxicity. The aim of the present study was to develop a method for the quantification of isobrucein B and neosergeolide in *P. sprucei* root and stem infusions based on reversed-phase high performance liquid chromatography (HPLC) and ultraviolet

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,

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,

(**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 structures.

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).

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

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

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 quassinoid composition of these infusions in the study on toxicity published by Fandeur *et al*. (1985), which focused only on the acute toxicity and antimalarial activity of isolated quassinoid components o *P. sprucei* and not on toxicity and antimalarial activity of infusions. Additional studies are needed to prove the *in vivo* efficacy and pharmacological activity of these infusions as antimalarials with focus on the dose-effect and dose-response to define the levels of toxicity. The aim of the present study was to develop a method for the quantification of isobrucein B and neosergeolide in *P. sprucei* root and stem infusions based on reversed-phase high performance liquid chromatography (HPLC) and ultraviolet detection (UV).
