**Use of Associated Chromatographic Techniques in Bio-Monitored Isolation of Bioactive Monoterpenoid Indole Alkaloids from** *Aspidosperma ramiflorum*

Talita Perez Cantuaria Chierrito1, Ananda de Castro Cunha1, Luzia Koike2, Regina Aparecida Correia Gonçalves1 and Arildo José Braz de Oliveira1 *1Department of Pharmacy, State University of Maringá, Maringá, Paraná State, 2State University of Campinas, Chemistry Institute, Campinas-SP, Brasil* 

#### **1. Introduction**

118 Chromatography and Its Applications

Yu-Tang Tung, Meng-Thong Chua, Sheng-Yang Wang, Shang-Tzen Chang, 2008. Anti-

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inammation activities of essential oil and its constituents from indigenous *Cinnamon* (*Cinnamomum osmophloeum*) twig. Bioresource Technology 99, 3908-

> The genus *Aspidosperma* (Apocynaceae) have been commonly used in folk medicine as potential antimalarial agents; in the treatment of leishmaniasis; uterus and ovary inflammations; as a contraceptive; in diabetes; stomach disorders; against cancer; fever and rheumatism (Oliveira et al, 2009). It commonly grows in tropical America, extracted from trees ranging 2 to 60 m in height. It is found in a variety of habitats from the dry fields of south-central Brazil, Paraguay, and Argentina to the inundated river margins of the Amazon basin (Tanaka, 2006). The main constituents of the *Aspidosperma* genus are indole alkaloids, a class of substances with a wide range of pharmacological activities such as cholinesterase inhibitors, analgesic, anti-inflammatory, bactericidal, oestrogenic, stimulant and depressant of the central nervous system (CNS) (Zocoler et al, 2005).

#### **1.1 Chromatographic systems in** *Aspidosperma* **genus**

Several methods are available for the analysis and identification of known indole alkaloids. Analysis of complex mixtures is frequently done by means of Thyn Layer Chromatography (TLC), through comparison of Rf values obtained in different solvent systems, and also by comparison between specific color reactions of components of the mixture and reference compounds. TLC remains one of the preferred methods for qualitative analysis of known compounds since it requires neither sophisticated equipment nor extensive sample preparation. For quantitative analysis, High Performance Liquid Chromatography (HPLC) systems linked to a UV detector are commonly used. By coupling HPLC to a photodiode array UV detector makes it possible to combine the information over retention times and the UV spectrum of each compound, and in some cases, it also enables the quantification of overlapping peaks. Capillary Gas Chromatography (GC) analysis has been described for several classes of alkaloids. A major advantage of GC over the above-mentioned methods is its enhanced sensitivity and high resolution. Another advantage is its easy coupling to a

Use of Associated Chromatographic Techniques in Bio-Monitored Isolation

**2. General methods of alkaloids extraction** 

against *S. aureus*.

of Bioactive Monoterpenoid Indole Alkaloids from *Aspidosperma ramiflorum* 121

for the activity against *Leishmania (L.) amazonensis* (Tanaka et al., 2007). Our results revealed that dimeric corynanthe alkaloids Ramiflorines A **(1)** and B **(2)** were responsible for the activity against promastigote forms of *L. amazonensis* with significant activity (LD50 values of 16.3 ± 1.6 µg/ml and 4.9 ± 0.9 µg/ml, respectively). Tanaka et.al. (2006) evaluated the antibacterial activities of the crude methanol extract, fractions obtained after acid-base extraction and pure compounds from the stem barks of *Aspidosperma ramiflorum* and both Ramiflorines showed significant activity against *S. aureus* (MIC = 25 µg/mL) and *E. faecalis* (MIC = 50 µg/mL), with EC50 of 8 and 2.5 µg/mL for Ramiflorines A and B, respectively,

From crude alcoholics or hidroalcoholics extracts obtained from parts of the plant material, two methods already described in the literature may be used for preliminary extraction of alkaloids; both methods take into account the basic nature of these compounds in order to concentrate them in pKas nearby and produce more purified fractions, preceding the phases of isolation and identification of substances. The first method consists of a complete acidbase partition (Figure 2), employing solvents such as chloroform or dichloromethane,

Fig. 2. Complete acid-base extraction from crude extract of *A. ramiflorum.* 

mass spectrometer that allows the identification of new and minor compounds of a mixture without laborious isolation procedures, which makes it a particularly attractive method when no decomposition due to the high temperatures applied in GC occurs (Dagnino, 1991). This chapter shows different chromatographic techniques (TLC, Preparative Thyn Layer Chromatography (PTLC), Classical Liquid Column Chromatography (CLCC), GC and HPLC) to isolate and characterize indole alkaloids of *Aspidosperma ramiflorum* species.

#### **1.1.2** *Aspidosperma ramiflorum* **species**

*Aspidosperma ramiorum* Muell. Arg. species commonly known as "guatambu", had been studied in 1996 by Reis et al., who were able to isolate the monoterpenoid indole alkaloids, ramiflorine A **(1)**, ramiflorine B **(2),** 10-methoxy-geissoschizol **(3)** and *β*-yohimbine (4). After that, Oliveira 1999 isolated beyond these compounds the 16-(*E*)-isositrikine **(5),** all them from stem barks (Figure 1).

Fig. 1. Major monoterpenoid indole alkaloids from *Aspidosperma ramiflorum*. Marvin was used for drawing, displaying and characterizing chemical structures, substructures and reactions, Marvin 5.4.1.1, 2011, ChemAxon available on ( http://www.chemaxon.com ).

The basic crude extract from stem barks of *A. ramiflorum* showed a good antileishmanial activity (Ferreira et al., 2004), which we attributed to the presence of indole alkaloids, and soon after, we described the fractionation, purification and isolation of alkaloids responsible for the activity against *Leishmania (L.) amazonensis* (Tanaka et al., 2007). Our results revealed that dimeric corynanthe alkaloids Ramiflorines A **(1)** and B **(2)** were responsible for the activity against promastigote forms of *L. amazonensis* with significant activity (LD50 values of 16.3 ± 1.6 µg/ml and 4.9 ± 0.9 µg/ml, respectively). Tanaka et.al. (2006) evaluated the antibacterial activities of the crude methanol extract, fractions obtained after acid-base extraction and pure compounds from the stem barks of *Aspidosperma ramiflorum* and both Ramiflorines showed significant activity against *S. aureus* (MIC = 25 µg/mL) and *E. faecalis* (MIC = 50 µg/mL), with EC50 of 8 and 2.5 µg/mL for Ramiflorines A and B, respectively, against *S. aureus*.
