Diterpenoids from the root extract of Chiococca alba

 

Eugenia V. Dzib-Reyes^a, Karlina García-Sosa^a, Paulino Simá-Polanco^b, Luis M. Peña-Rodríguez^a,*

 

^a Unidad de Biotecnología. *Corresponding autor. Tel.: +52-999-9428330 ext. 159; Fax: +52-999-9813900; email: lmanuel@cicy.mx.

^b Unidad de Recursos Naturales, Centro de Investigación Científica de Yucatán, Calle 43 No. 130, Col. Chuburná de Hidalgo, 97200 Mérida, Yucatán, México.

 

Received September 2012.
Accepted October 2012.

 

ABSTRACT

Two new ent-kaurane diterpenes, 1-hydroxy-18-nor-kaur-4,16-dien-3-one (1) and 15-hydroxy-kaur-16-en-3-one (2), along with the four known metabolites kaur-16-en-19-ol (3), kaurenoic acid (4), merilactone (5), and ribenone (6), and a mixture of stigmasterol and β-sitosterol were isolated from the ethanolic root extract of Chiococca alba. Chemical structures of the different metabolites were established through the interpretation of their spectroscopic data and/or by comparing them with those reported in the literature.

Keywords: Rubiaceae, Chiococca alba, kaurane, pimarane, labdane, diterpenes.

 

RESUMEN

Dos nuevos diterpenos tipo ent-kaurano, 1-hidroxi-18-nor-kaur-4,16-dien-3-ona (1) y 15-hidroxi-kaur-16-en-3-ona (2), además de cuatro metabolitos ya conocidos, kaur-16-en-19-ol (3), ácido kaurenoico (4), merilactona (5), y ribenona (6), y una mezcla de estigmasterol y β-sitosterol, fueron aislados del extracto etanólico de la raíz de Chiococca alba. Las estructuras químicas de los diferentes metabolitos fueron establecidas mediante la interpretación de sus datos espectroscópicos y/o por comparación con los datos reportados en la literatura.

Palabras clave: Rubiaceae, Chiococca alba, kaurano, labdano, pimarano, diterpenos.

 

INTRODUCTION

Chiococca alba (L.) Hitchc. (Rubiaceae), commonly known as "t'unché" or "kanchakché" in the Yucatán peninsula (Mendieta and Del Amo, 1981), is a tropical and sub-tropical shrub distributed all over the American continent (Standley and Williams, 1975). The root decoction of this plant is used to cure dysentery, as a diuretic and cathartic, and against snake bites (Mendieta and Del Amo, 1981; Argueta et al., 1994), while the leaf infusion is used to alleviate various ailments such as asthma, headaches and diarrhea, and a decoction of the whole plant is reported to be effective as a laxative, against gonorrhea, to cure skin infections, and to treat rheumatism (Mendieta and Del Amo, 1981; Del Amo, 1980). Although the Chiococca genus includes more than twenty species found throughout the American continent (Lopes et al., 2004), its phytochemical and pharmacological knowledge is limited to a number studies carried out on C. alba which have reported the isolation of alkaloids (El Abbadi et al., 1989), flavonoids (Lopes et al., 2004), iridoids (Carbonezi et al., 1999), saponins (Borges et al., 2009), and terpenoids (Lopes et al., 2004; Carbonezi et al., 1999; Borges et al., 2009; Bhattacharyya and Cunha, 1992; Borges-Argaez et al., 1997, 2001) from the roots of the plant, and lignans, coumarins and ketoalcohols from the leaves (El Hafiz et al., 1991). As part of our search for novel metabolites from medicinal plants of the Yucatan peninsula, we wish to report herein on the isolation and identification of the novel ent-kauranes 1-hydroxy-18-nor-kaur-4, 16-dien-3-one (1) and 15-hydroxy-kaur-16-en-3-one (2), together with the known diterpenes kaur-16-en-19-ol (3), kaurenoic acid (4), merilactone (5) and ribenone (6), and a mixture of stigmasterol and p-sitosterol, from the root extract of C. alba.

 

EXPERIMENTAL

General experimental procedures.
Analytical and preparative TLC were carried out using precoated Si gel aluminum and glass plates respectively (Merck 60F₂₅₄, 0.25 and 0.50 mm thickness). Chromatograms were examined under UV light in a UV-viewing cabinet and visualized by dipping in 4% phosphomolybdic acid solution containing a trace of ceric sulfate in 5% sulfuric acid, followed by drying and gentle heating. GC/MS analyses were performed on an Agilent Technologies 6890N gas chromatograph [1.0 μL of 3% sample in CHCl₃, HP 5MS (30 m x 0.25 mm i.d. x 30 μm), flow rate 1mL/min, temperature program: T₁ 150 °C, T₂ 280 °C, gradient 10 °C/min, injector temperature 280 °C, detector temperature 280 °C] coupled to an Agilent Technologies 5975B mass selective detector. IR spectrum was recorded in CHCl₃ (film) using a FT-IR Nicolet Magna 750 spectrophotometer, and ¹H-NMR (400 MHz) and ¹³C-NMR (100 MHz) spectra were obtained on a Bruker Avance 400, Variant Unity Plus-300 and Bruker DRX400, using the residual CHCl₃ signal (7.26 and 77.00 ppm for ¹H and ¹³C, respectively) as reference.

Plant material. The roots of C. alba were collected during march 2008 and 2009, from plants growing in a field located at km 7 of Yaxcabá higway in Yucatán, México. A voucher specimen has been deposited in the herbarium of the Unidad de Recursos Naturales of the Centro de Investigación Científica de Yucatán (CICY) under the collection number 3200. The plant material was washed with tap water and dried, first for a week at room temperature, and then for 72 h in an oven at 50 °C. The dried roots were ground using a Pagani Dycomet 1520 mill and a No. 6 sieve.

Extraction and isolation. The dryroots (3.95 Kg) were extracted four times with EtOH (6 L) at room temperature. The extracts were combined and the solvent was removed under reduced pressure to produce 209.5 g of crude ethanolic extract. The crude extract was suspended in 1725 ml of a 3:2 mixture of H₂O:MeOH and the resulting aqueous suspension was subjected to a liquid-liquid partition procedure with hexane and ethyl acetate to produce the corresponding low (19.5 g) and medium (55.6 g) polarity fractions, respectively. Successive purifications of the low polarity fraction, using a combination of vacuum liquid chromatography, flash column chromatography, gravity column chromatography and preparative-TLC, led to the isolation of metabolites 1 (45.5 mg), 2 (3.0 mg), 3 (22.3 mg), 4 (10.9 mg), 5 (11.6 mg), and 6 (2.1 mg) in pure form, and 3.7 mg of an inseparable mixture of stigmasterol and β-sitosterol.

1-hydroxy-18-nor-kaur-4, 16-dien-3-one (1), colorless oil, soluble in CH₂Cl₂, single spot on TLC visible under UV light, R_f=0.48 (Et₂O/Hx 6:4); [α]²⁵_D= -20.0 (c 0.0116, CHCl₃); GC-MS: t_R= 12.9 min, m/z 286.2 [M⁺]; IR (CHCl₃ 2%): 3477.7, 1669.9,1613.7 cm^{-1 1}H-NMR (CDCl₃, 400 MHz): see data in Table 1.

15-hydroxy-kaur-16-en-3-one (2), obtained as a crystalline solid, soluble in CH₂Cl₂, R_f=0.37 (Et₂O/Hx 6:4); GC-MS: t_R=14.0 min, m/z 302.2 [M⁺]; IR (CHCl₃): 3457.0, 1705.8 cm¹. ¹H-NMR (CDCl₃, 400 MHz): 1.55 (H-1a, m, 1H); 2.02 (H-1b, ddd, 3.9, 6.4, 13.0, 1H); 1.53 (H-2, m, 2H); 1.45 (H-5/6a, m, 2H); 1.64 (H-6b, m, 1H); 2.48 (h-7, ddd, 3.6, 6.4, 8.8, 2H); 1.13 (H-9, d, 7.9, 1H); 1.64 (H-11, m, 2H); 1.43 (H-12a, d, 2.5, 1H); 1.79 (H-12b, dd, 3.3, 8.7, 1H); 2.78 (H-13, bs, 1H), 1.40 (H-14a, d, 4.0, 1H); 1.87 (H-14b, dd, 1.4, 11.8, 1H); 3.82 (H-15, bs, 1H); 5.10 (H-17a, bs, 1H); 5.23 (H-17b, bs, 1H); 1.10 (H-18, s, 3H); 1.05 (h-19, s, 3H); 1.08 (H-20, s, 3H); ¹³C-NMR (CDCl₃, 100 MHz): 39.4 (C-1, t); 32.6 (C-2, t); 207.3 (C-3, s); 47.2 (C-4, s); 54.4 (C-5, d); 20.8 (C-6, t); 34.1 (C-7, t); 47.6 (C-8, s); 53.0 (C-9, d); 38.7 (C-10, s); 18.7 (C-11, t); 34.2 (C-12, t); 42.3 (C-13, d); 36.0 (C-14, t); 82.6 (C-15, d); 160.0 (C-16, s); 108.7 (C-17, t); 27.5 (C-18, q); 21.1 (C-19, q); 18.1 (C-20, q) ppm.

 

RESULTS AND DISCUSSION

The ethanolic root extract of C. alba was first fractionated using a liquid-liquid partition procedure with hexane and ethyl acetate. Purification of the resulting low-polarity (hexane) fraction using different chromatographic techniques produced diterpenes 1 -6 in pure form, together with a mixture of stigmasterol and β-sitosterol.

The molecular formula of 1 , established as C₁₉H₂₆O₂ from its MS data (parent ion peak at m/z 286.2), implied the presence of seven unsaturation sites in its structure. The IR spectrum of 1 exhibited strong absorption bands at 3477 and 1669 cm^-1, indicating that the two oxygen atoms in the molecular formula were in the form of hydroxyl and carbonyl groups, respectively. The presence of these two functional groups in the structure of 1 was further supported by the signals at δ 200.5 and 68.2 in its ¹³C-NMR spectrum (Table 1), while the four additional sp²-carbon signals at δ 154.4, 104.6, 166.8, 124.0, corresponding to the carbon atoms of two double bonds, strongly suggested that the four remaining unsaturation sites were in the form of a tetracyclic structure. The combined analysis of the ¹³C-NMR spectrum of 1 , together with the data from its HSQC, DEPT-135 and DEPT-90 experiments, allowed the identification of all carbon atoms in the structure and indicated the presence of two methyl groups, eight methylenes, three methines and six quaternary carbons.

The ¹H-NMR spectrum of 1 (Table 1) showed the presence of an axial-oriented carbinol proton at δ_H 4.09 (ddd, J=13.8, 6.1, 2.0 Hz), together with two vinylic protons of an exocyclic double bond at δ_H 4.81 and 4.86, and a vinylic methyl group at δ_H 1.85. In the ¹H-¹H COSY experiment, the clear correlation observed between the carbinol proton at δ_H 4.09 and the signals corresponding to two methylene protons at δ_H 2.53 (dd, J=13.8, 6.1 Hz) and δ_H 1.34 (d, J= 13.9 Hz) indicated their being an isolated system. However, a detailed analysis of the HMBC experiment (Table 1) allowed the construction of the full structure; the ²J and ³J correlations observed between the protons of the methylene group at δ_H 2.53/1.34 and the carbonyl carbon at δ_C 200.5 and the sp² carbon at δ_C 166.8, respectively, together with the ³J and ²J correlations observed between these two carbons and the vinylic methyl group at δ_H 1.85, respectively, and the ³J correlation observed between the carbinol proton at δ_H 4.09 and the methyl group at δ_C 27.4 indicated that they were all located in the same ring. Similar correlations allowed the construction of the remaining tetracyclic structure; a through literature search indicated that metabolite 1 is a new natural product with a nor-diterpene ent-kaurane skeleton.

Metabolite 2 showed a parent ion peak at m/z 302.2 in its mass spectrum, which indicated a molecular formula of C₂₀H₃₀O₂ that, in turn, implied the presence of six unsaturation sites in the structure. The IR spectrum of 2 exhibited a strong hydroxyl group absorption band at 3457 cm^-1 and another one in the carbonyl region at 1705 cm^-1, indicating that, as in the case of 1, the two oxygen atoms in the molecular formula were in the form of hydroxyl and carbonyl groups, respectively; this was confirmed by the signals corresponding to a carbonyl and to an sp³-oxygenated carbon at δ_C 207.3 and 82.6, respectively, in the ¹³C-NMR spectrum of 2. Additionally, the signals corresponding to the two sp²-carbons of an exocyclic double bond at δ_C 160.0, 108.7 (Wehrli and Nishida, 1979), identified the second insaturation in the structure and suggested that the four remaining unsaturation sites corresponded to a tetracyclic ent-kaurane diterpenoid structure. The nature of all carbon atoms was confirmed through a combined analysis of the ¹³C-NMR spectrum and the HSQC experiment and indicated the expected presence of three methyl groups, nine methylenes, three methines and five quaternary carbons. The ¹³C and ¹H chemical shift values of both protons and carbons were compared to those reported in the literature (Wehrli and Nishida, 1979; Baptista et al., 2007; Pacheco et al., 2009); the search indicated that metabolite 2 represents a new natural ent-kaurane.

Metabolites 3, 4, 5 and 6 were found to be identical with kaur-16-en-19-ol (Wehrli and Nishida, 1979; Baptista et al., 2007; Pacheco et al., 2009), kaurenoic acid (Pacheco et al., 2009; Da Silva et al., 2001; Dang et al., 2005; Batista et al., 2007; La-cruz, 2008; Lee, et al., 2008), merilactone (Borges-Argaez et al., 2001), and ribenone (wherli and Nishida, 1979; Da Silva et al., 2001; Almquist, et al., 1975; Konishi et al., 1996; Wantanee et al., 2003), by comparing their spectroscopic data (MS, IR, ¹H and ¹³C NMR) with those reported in the literature. Although metabolites 3, 4 and 6 have been found in several plant families such as Euphorbiaceae, Aristolochiaceae and Astaraceae, respectively, this is the first report of their occurrence in the Rubiaceae family and in C. alba in particular. Merilactone (5) is the only metabolite that has been previously isolated from the root extract of the plant.

 

CONCLUSIONS

The results obtained in this investigation confirm the root extract of Chiococca alba as an important source of novel diterpenoids, particularly of those having labdane, pimarane and ent-kaurane skeletons.

 

ACKNOWLEDGEMENTS

EVDR gratefully acknowledges the support of The American Society of Pharmacognosy (ASP), through their Undergraduate Research Award Programme, which enabled her to work on this project. This work was supported by Project FOMIX-Yucatán (66262).

 

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Note

This paper is dedicated to Professor Pedro Joseph-Nathan in recognition of his 50 years of outstanding scientific trajectory.