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


Introduction
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 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 Quantification of Antimalarial Quassinoids Neosergeolide and Isobrucein B 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 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, IC 50 =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. 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 (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 (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 www.intechopen.com Quantification of Antimalarial Quassinoids Neosergeolide and Isobrucein B 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 detection (UV).

Reagents and solvents
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).

www.intechopen.com
Chromatography and Its Applications 192

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.

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.

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.

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.

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 (t i =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 (t f =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.

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). 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.
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.

Results and discussion
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 1 H and 13 C of 1 and 2 are presented in tables 1 and 2 respectively. 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.

Root infusion
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.

Conclusion
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 plays more important role for the antimalarial activity than neosergeolide.
More research is needed to describe seasonal, regional and specimen specific variation in P. sprucei quassinoid composition which should have a direct influence on the composition of stem and root infusions prepared from samples of different origins. Knowledge of the extent of these variations, especially as they influence quassinoid composition in infusions, is of fundamental importance given the valuable medicinal and dangerous toxic properties of these widely used Amazonian remedies.
The high performance liquid chromatography has proved to be a powerful tool in the plant extract analysis. The possibility to perform qualitative and quantitative analysis, by HPLC, enables the development of new phytoterapeutical products from the Amazon biodiversity.

Acknowledgment
The autors wish to thank Dr Wanderli Pedro Tadei and CNPq/ FAPEAM -PRONEX, Rede Malária for financial support to the publication of this chapter, Massuo Kato for use of analytical HPLC apparatus and Profs. Norberto P. Lopes and Valquiria P. Jabor for helpful comments regarding this manuscript.