Chemical constiuents from Richardia grandiflora (Cham. & Schltdl.) Steud. (Rubiaceae)

Constituintes químicos de Richardia grandiflora (Cham. & Schltdl.) Steud. (Rubiaceae)

Abstracts

Amongst the different forms of therapy to prevent and cure illnesses, plants have been, undoubtedly, the most utilized ones since the beginning of mankind. Brazil has a great diversity on plants that possess non-researched medicinal potential and are promising sources of therapeutic and pharmacological innovations. The Rubiaceae family is considered the biggest one of the order Gentianales, presenting around 637 genera and 10,700 species. Richardia grandiflora (Cham. & Schltdl.) Steud., known popularly as "ervanço", "poaia" or "ipeca-mirim", has ethnopharmacological indications to use as decoction against hemorrhoids and as vermifuge. Aiming at contributing to the chemotaxonomic study of the family Rubiaceae and considering the absence of data in literature about the chemical constitution of the species Richardia grandiflora, the latter was submitted to a phytochemical study to isolate its chemical constituents, through usual chromatographic methods, and after identifying them by means of spectroscopic methods such as ¹H and 13C NMR, with the add of two-dimensional techniques, besides comparison with literature data. Five constituents were isolated through this first phytochemical study with R. grandiflora: a mixture of the steroids beta-sitosterol and stigmasterol, o-hydroxy-benzoic acid, m-methoxy-p-hydroxy-benzoic acid and phaeophitin A, all of them isolated for the first time from the genus Richardia.

Richardia grandiflora; Rubiaceae; beta-sitosterol; stigmasterol; o-hydroxy-benzoic acid; m-methoxy-p-hydroxy-benzoic acid; phaeophitin A

Dentre as diversas formas de terapia para a prevenção e cura de doenças, as plantas foram, indubitavelmente, as mais amplamente utilizadas desde o início da humanidade. O Brasil tem grande diversidade de plantas com potenciais medicinais, ainda não pesquisados, e que são promissoras fontes de inovações terapêuticas e farmacológicas. A família Rubiaceae, considerada a maior da ordem Gentianales, possui cerca de 637 gêneros e 10.700 espécies. Richardia grandiflora (Cham. & Schltdl.) Steud., conhecida popularmente como ervanço, poaia ou ipeca-mirim, tem indicações etnofarmacológicas para uso contra hemorróidas e como vermífugo na forma de decocto. Visando a contribuir com o estudo quimiotaxonômico da família Rubiaceae e tendo em vista a ausência de dados na literatura acerca da constituição química de Richardia grandiflora, esta foi submetida a um estudo fitoquímico para o isolamento de seus constituintes químicos, através dos métodos cromatográficos usuais, e posterior identificação estrutural dos mesmos, utilizando-se os métodos espectroscópicos de RMN ¹H e 13C uni e bidimensionais, além de comparações com modelos da literatura. Deste estudo pioneiro com R. grandiflora foram isolados e identificados cinco constituintes: uma mistura dos esteróides beta-sitosterol e estigmasterol, o ácido o-hidroxibenzóico, o ácido m-metoxi-p-hidroxi-benzóico e a feofitina A, todos inéditos no gênero Richardia.

Richardia grandiflora; Rubiaceae; beta-sitosterol; estigmasterol; ácido o-hidroxibenzóico; ácido m-metoxi-p-hidroxi-benzóico; feofitina A

ARTIGO

Chemical constiuents from Richardia grandiflora (Cham. & Schltdl.) Steud. (Rubiaceae)

Constituintes químicos de Richardia grandiflora (Cham. & Schltdl.) Steud. (Rubiaceae)

Anna Cláudia de A. Tomaz^*; Raquel Bezerra S. S. Nogueira; Danielle Serafim Pinto; Maria de Fátima Agra; Maria de Fátima V. de Souza; Emídio V. Leitão da-Cunha

Laboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, Caixa Postal 5009, 58051-970 João Pessoa-PB, Brazil

ABSTRACT

Amongst the different forms of therapy to prevent and cure illnesses, plants have been, undoubtedly, the most utilized ones since the beginning of mankind. Brazil has a great diversity on plants that possess non-researched medicinal potential and are promising sources of therapeutic and pharmacological innovations. The Rubiaceae family is considered the biggest one of the order Gentianales, presenting around 637 genera and 10,700 species. Richardia grandiflora (Cham. & Schltdl.) Steud., known popularly as "ervanço", "poaia" or "ipeca-mirim", has ethnopharmacological indications to use as decoction against hemorrhoids and as vermifuge. Aiming at contributing to the chemotaxonomic study of the family Rubiaceae and considering the absence of data in literature about the chemical constitution of the species Richardia grandiflora, the latter was submitted to a phytochemical study to isolate its chemical constituents, through usual chromatographic methods, and after identifying them by means of spectroscopic methods such as ¹H and ¹³C NMR, with the add of two-dimensional techniques, besides comparison with literature data. Five constituents were isolated through this first phytochemical study with R. grandiflora: a mixture of the steroids b-sitosterol and stigmasterol, o-hydroxy-benzoic acid, m-methoxy-p-hydroxy-benzoic acid and phaeophitin A, all of them isolated for the first time from the genus Richardia.

Keywords:Richardia grandiflora, Rubiaceae, b-sitosterol, stigmasterol, o-hydroxy-benzoic acid, m-methoxy-p-hydroxy-benzoic acid, phaeophitin A.

RESUMO

Dentre as diversas formas de terapia para a prevenção e cura de doenças, as plantas foram, indubitavelmente, as mais amplamente utilizadas desde o início da humanidade. O Brasil tem grande diversidade de plantas com potenciais medicinais, ainda não pesquisados, e que são promissoras fontes de inovações terapêuticas e farmacológicas. A família Rubiaceae, considerada a maior da ordem Gentianales, possui cerca de 637 gêneros e 10.700 espécies. Richardia grandiflora (Cham. & Schltdl.) Steud., conhecida popularmente como ervanço, poaia ou ipeca-mirim, tem indicações etnofarmacológicas para uso contra hemorróidas e como vermífugo na forma de decocto. Visando a contribuir com o estudo quimiotaxonômico da família Rubiaceae e tendo em vista a ausência de dados na literatura acerca da constituição química de Richardia grandiflora, esta foi submetida a um estudo fitoquímico para o isolamento de seus constituintes químicos, através dos métodos cromatográficos usuais, e posterior identificação estrutural dos mesmos, utilizando-se os métodos espectroscópicos de RMN ¹H e ¹³C uni e bidimensionais, além de comparações com modelos da literatura. Deste estudo pioneiro com R. grandiflora foram isolados e identificados cinco constituintes: uma mistura dos esteróides b-sitosterol e estigmasterol, o ácido o-hidroxibenzóico, o ácido m-metoxi-p-hidroxi-benzóico e a feofitina A, todos inéditos no gênero Richardia.

Unitermos:Richardia grandiflora, Rubiaceae, b-sitosterol, estigmasterol, ácido o-hidroxibenzóico, ácido m-metoxi-p-hidroxi-benzóico, feofitina A.

INTRODUCTION

Amongst the different forms of therapy to prevent and cure illnesses, plants have been, undoubtedly, the most utilized ones since the beginning of mankind. Plants were originally used in their natural form in the preparation of teas, ointments, plasters and others. Later, especially from the beginning of the XIX century, they served as source for obtaining raw material for the synthesis of drugs. More recently, plants have emerged as keys to the discovery of prototypes that serve as rational basis to the development of medicines (Funari and Ferro, 2005; Albuquerque et al., 2006; Vilegas and Cardoso, 2007)

Brazil has a great diversity on plants that possess non-researched medicinal potential and are promising sources of therapeutic and pharmacological innovations to the most diverse areas of human health (Almeida et al., 2001; Silva et al., 2003; Rocha et al., 2005; Brandão et al., 2006; Carlini et al., 2006; Silva et al., 2006a;). The medicinal, economical and ecological importance of Brazilian native species, as well as the risk of their extinction by man’s predatory action, has been motivating the studies on these plants, seeking their preservation and rational exploitation (Souza et al., 2003; Barbosa-Filho et al., 2005; Falcão et al., 2005; Amaral et al., 2006; Barbosa-Filho et al., 2006a,b,c; Leitão et al., 2006; Lima et al., 2006; Barbosa-Filho et al., 2007; Oliveira et al., 2007; Saúde-Guimarães, et al., 2007; Rocha et al., 2007).

The Rubiaceae family is considered the biggest one of the order Gentianales (Coelho et al., 2006) and presents around 637 genera and 10,700 species (Mongrand et al., 2005). It is composed of plants of varied habit, is cosmopolitan, its species are classified into four subfamilies (Cinchonoideae, Ixoroideae, Antirheoideae e Rubioideae) and 44 tribes, mostly tropical (Robbrecht, 1988).

Rubiaceae has species of great economical importance which are exploited as food, ornamental and in the pharmaceutical industry too (Carvalho et al., 2006). In addition, several species are popularly referred to as toxic and/or medicinal and among these latter there are species of the genus Richardia (Coelho et al., 2006).

Richardia is a genus of about 15 species in the family Rubiaceae, mainly distributed from North to South America. They are annual or more commonly perennial herbs (Lewis and Oliver, 1974).

Species of this genus are reported as weeds (Hauser and Parham, 1969; Monquero, 2003; Monquero and Christoffoleti, 2003; Ronchi et al., 2003; Pedrinho Júnior et al., 2004; San Martin Matheis, 2004; Monquero, 2005; Monquero et al., 2005) and recognize them is important in order to benefit the agricultural planning.

Richardia grandiflora (Cham. & Schltdl.) Steud. is native to Argentina, Bolivia, Brazil, Paraguay and Uruguay (Lewis and Oliver, 1974). According to Agra et al. (2007), this species is known popularly as "ervanço", "poaia" or "ipeca-mirim", and has ethnopharmacological indications to use as decoction against hemorrhoids and as vermifuge.

There are no data in literature concerning the chemical constitution of the species in question. However, Edeoga et al. (2005) reported the presence of alkaloids, tannins, saponins, steroids, terpenoids, flavonoids and cardiotonic glycosides through preliminary phytochemical screening in Richardia brasiliensis Gomes and reported that it is used in folk medicine to cure eczema, in the treatment of burns and in the active cure to avian malaria. The water and ethanol extracts of this species showed antifungal activity observed by Adekunle (2000).

Therefore, aiming at contributing to the chemotaxonomic study of the family Rubiaceae and considering the absence of data in literature about the chemical constitution of Richardia grandiflora, the latter was submitted to a phytochemical study to isolate and identify its chemical constituents, through usual chromatographic and spectroscopic methods, besides comparison with literature data.

MATERIAL AND METHODS

Equipments

NMR spectra (¹H, ¹³C, HMQC, HSQC, HMBC, COSY, NOESY) were run on a Mercury Varian instrument operating at 200 MHz (¹H ) and 50 MHz (¹³C) (LTF/UFPB) (substances 1, 2, 4 and 5) and on a BRUKER-AC operating at 500 MHz (¹H ) and 125 MHz (¹³C) (CENAUREM) (substance 3). The NMR data were measured in CDCl₃ and the chemical shifts are expressed in ppm with reference to the solvent signal.

Plant material

Richardia grandiflora (Cham. & Schltdl.) Steud. was collected in the city of João Pessoa, State of Paraíba, being identified by Prof. Maria de Fátima Agra from the Botany section of Núcleo de Pesquisa de Produtos Naturais of Laboratório de Tecnologia Farmacêutica (LTF). A voucher specimen is deposited in the Herbarium Professor Lauro Pires Xavier (JPB) from Centro de Ciências Exatas e da Natureza of Universidade Federal da Paraíba (UFPB) under the code M. F. Agra et al. 2953 (JPB).

Extraction, fractionation and isolation of the chemical constituents

The plant material was subjected to dehydration in an oven in a temperature of 40 ºC for 72 hours; after that, it was grownded in a mechanical mill, yielding 3.164 kg of a powder which was submitted to maceration with ethanol for three consecutive days. This process was repeated until the maximum extraction of the chemical constituents. The obtained ethanol extractive solution was concentrated in a rotatory evaporator, yielding 400 g (12.64% yield in relation to the dried plant weight) of crude ethanol extract (CEE).

An aliquot of 100 g of the CEE was subjected to filtration under reduced pressure using silica gel 60 as stationary phase into a funnel of porous plate and was eluted with various systems of solvents (pure or in binary mixtures): Hexane, Hex:AcOEt (9:1), Hex:AcOEt (7:3), Hex:AcOEt (1:1), AcOEt:Hex (6:4), AcOEt:Hex (7:3), AcOEt:Hex (9:1), AcOEt, AcOEt:MeOH (9:1) and AcOEt:MeOH (7:3), yielding the respective fractions.

The fraction Hex:AcOEt (7:3) (1.238 g) was subjected to column chromatography packed with silica gel 60 and eluted with hexane, ethyl acetate and methanol; 278 fractions of 50 mL were colected being analysed and joined through analytical thin-layer chromatography (TLC). The sub-fraction 32/76, after being recrystallized in hexane showed itself as crystals, yielding 23 mg of the substance 1. The sub-fraction 104/149 (118 mg) was submmited to column chromatography over silica gel 60, giving 137 fractions that were analysed and joined through analytical TLC. The sub-fraction 87/120 (60 mg) showed itself as dark-green amorphous solid, defined as substance 3.

The fraction AcOet:Hex (9:1) (0.569 g) was subjected to column chromatography packed with Sephadex LH 20 and eluted with chloroform and methanol, from which 32 fractions of 15 mL were colected and then analysed and joined through analytical TLC. The sub-fraction 15/26 (6 mg), after being recrystallized with chloroform showed itself as crystals, yielding the substance 4.

The fraction Hex:AcOet (1:1) (1.895 g) was subjected to chromatography on a column packed with silica gel 60, the eluents were chloroform, ethyl acetate and methanol, giving 128 fractions of 50 mL that were analysed and joined through analytical TLC. The sub-fraction 103/104 (182 mg) was submitted to column chromatography packed with silica gel 60 and eluted with hexane, ethyl acetate and methanol, yielding 78 fractions of 50 mL that were analysed and joined through analytical TLC. The sub-fraction 35/39, after being recrystallized in hexane showed itself as crystals, yielded 4 mg of the substance 5.

RESULTS AND DISCUSSION

Structural characterization of the substances 1, 4 and 5

The spectral data together with comparison with literature data permitted to identify the substance 1 as a mixture of the steroids b-sitosterol (1) and stigmasterol (2) (Kojima et al., 1990), the substance 4 as the o-hydroxy-benzoic acid, and the substance 5 as the m-methoxy-p-hydroxy-benzoic acid (Figure 1). The ¹H and ¹³C NMR (d, CDCl₃, 200 and 50 MHz) spectral data of 4 and 5 are listed below.

o-Hydroxy-benzoic Acid (4). ¹H NMR (200 MHz, CDCl₃) d_H (H; mult.; J in Hz.): 6.98 (H-3; ld; 7.95), 7.49 (H-4; td; 7.95 and 1.50), 6.90 (H-5; lt; 7.90), 7.90 (H-6; dd; 7.90 and 1.50). ¹³C NMR (50 MHz, CDCl₃) d_C: 111.14 (C-1), 162.14 (C-2), 117.73 (C-3), 136.75 (C-4), 119.49 (C-5), 130.85 (C-6), 174.29 (COOH). All the values are according to literature data (Silva et al., 2006b).

m-Methoxy-p-hydroxy-benzoic Acid (5). ¹H NMR (200 MHz, CDCl₃) d_H (H; mult.; J in Hz): 7.57 (H-2; d; 1.60), 6.95 (H-5; d; 8.50), 7.72 (H-6; dd; 8.50 and 1.60), 3.94 (OCH₃; s). ¹³C NMR (50 MHz, CDCl₃) d_C: 121.180 (C-1), 112.04 (C-2), 146.16 (C-3), 150.76 (C-4), 114.18 (C-5), 125.16 (C-6), 171.05 (C-7), 56.10 (OCH₃). All the values are according to literature data (Costa et al., 2007).

Structural characterization of 3

The ¹H NMR spectrum revealed absorptions referable to three olefinic methyl groups at d_H 3.19, d_H 3.39 and d 3.69, a vinyl group at d_H 7.95 (dd, J 17.85 Hz and 11.48 Hz), d_H 6.27 (d, J 17.95 Hz), d_H 6.18 (d, J 11.10 Hz) and three olefinic hydrogens at d_H 9.35, d_H 9.51 and d_H 8.60 which are related to the absorptions of the hydrogens 5, 10 and 20 of the porphyrin skeleton of phaeophytins (Matsuo et al., 1996; Duan et al., 2002; Silva et al., 2006b). An ethyl group was found to occur at C-8 according to the absorptions at H-8² 3.63 (m) and H-8¹ 1.66 (m). The methoxyl hydrogens were observed as a singlet at d_H 3.91.

Similarly, as with phaeophytin A, a five membered carbocyclic ring was present at position 13. A keto group was found in C-13¹, as indicated by the quartenary carbon resonance at d_C 189.81. The H-13² resonance occurred at d_H 6.30 (s) as with phaeophytin at and a methyl ester was also present at C-13³. This was indicated by C-13³ occurring as a quaternary carbon resonance at d_H 169.77 and the C-13⁴ methyl carbon resonance being present at d_C 53.07. Nevertheless, the chemical shift revealed by the ¹³C NMR spectrum to the carbonyl carbon at the position 13³ is not in agreement with the literature data. Matsuo et al. (1996) and Schwikkard et al. (1998) report that this value is of d_C 173.0. Our attribution is unambiguous since the heteronuclear correlation spectrum (HMBC) showed a three-bond correlation (³J_CH) between the carbon 13³ (d_C 169.77) and the hydrogen 13⁴ (d_H 3.91), and a two-bond correlation (²J_CH) between the carbon 13³ and the hydrogen 13² (d_H 6.30), thus strengthening our data.

Phaeophytin A is substituted with a phytyl ester at C-17³ (Schwikkard et al., 1998). The presence of an envelope of signals in the aliphatic region of the ¹H NMR spectrum of 3 and the resonance found to C-17³ (d_C 173.18) suggests the presence of a phytyl ester in the molecule when compared with the data of the 17³-ethoxy-pheophorbide A (Silva et al., 2006b) which does not have the phytyl.

The COSY spectrum confirmed the presence of vinyl and ethyl groups showing coupling between d_H 7.95 (H-3¹) and d_H 6.27 (H-3²) (E), d_H 6.18 (H-3²) (Z), referable to vinyl hydrogens, and between d_H 3.63 (H-8¹) and d_H 1.66 (H-8²) the ethyl hydrogens.

The other assignments of carbons and hydrogens were determined based on all spectral data (Table 1) and on comparison with literature data (Matsuo et al., 1996; Schwikkard et al., 1998; Duan et al., 2002; Silva et al., 2006b), permitting to identify the substance 3 as being phaeophytin A (Figure 2), substance described for the first time in the genus Richardia.

ACKNOWLEDGMENTS

The authors wish to express their thanks to CAPES and CNPp and IMSEAR for financial support and to CENAUREM for providing the spectra of phaeophytin A.

Received 7 August 2007; Accepted 12 December 2007

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Publication Dates

• Publication in this collection
  14 Apr 2008
• Date of issue
  Mar 2008

History

• Received
  07 Aug 2007
• Accepted
  12 Dec 2007