Constituents of Corynaea crassa "Peruvian Viagra"

Abstract

A phytochemical investigation of methanol and n-hexane extracts of tuber/roots of Corynaea crassa Hook. f., Balanophoraceae, led to the isolation and characterization of β-sitosterol, lupenone, β-amyrone, lupeol, and β-amyrine. Unusual complex 1:1 mixtures of lupenone/β-amyrone and lupeol/β-amyrine obtained from the extracts were identified by NMR and HR-MS experiments. The structure of the 1:1 lupenone/β-amyrone mixture was confirmed by X-ray analysis. These triterpene ketone derivatives, only distinguished either by 5- or 6-membered E ring, co-crystallize in one common unit cell in the solid state.

Keywords:
β-Amyrone; β-Amyrine; Corynaea crassa ; Huanarpo; Lupenone; Lupeol/β-sitosterol

Introduction

Medicinal plants with aphrodisiac activity are used for the treatment of erectile dysfunction all over the world. A variety of botanicals such as Tribulus terrestris L., Zygophyllaceae, Aframomum melegueta (Roscoe) K. Schum., Zingiberaceae, Eurycoma longifolia Jack, Simaroubaceae, Cnidium monnieri (L.) Cusson, Apiaceae, Ferula hermonis Boiss., Apiaceae, Mucuna pruriens (L.) DC., Fabaceae, Lepidium meyenii Walp., Brassicaceae, Passiflora incarnata L., Passifloraceae, and some compounds like yohimbine were reported to have beneficial effects on sexual function, supporting older claims and offering new hope (Hosseinzadeh et al., 2008).

Likewise, in contemporary Peruvian folk medicine a very large number of medicinal plants are used for aphrodisiacal purposes, a few hundred of them to modulate fertility, or to induce sterility (Pachacuti-Yamqui, 1992). Plant mixtures and magic are commonly applied to increase or to decrease libido (Elferink, 2000). One of the plants used in these aphrodisiac mixtures is commonly known as Chutarpo (or huanarpo), for which there is a female and male designation. Jatropha macrantha Müll. Arg., Euphorbiaceae (Bussmann and Sharon, 2007; Pardo, 2002; De Feo, 1992; Brack Egg, 1999; Desmarchelier et al., 1996a,b; Oshima et al., 2003; Tinco et al., 2011; Benavides et al., 2006; Okuyama et al., 1996; Schultes, 1980) and Corynaea crassa Hook. f., Balanophoraceae (Bussmann and Sharon, 2006, 2007; Bussmann and Glenn, 2010) are both under the male designation "Huanarpo macho". "Huanarpo macho" is traditionally used strictly as a male aphrodisiac, while the "Huanarpo hembra" Cnidoscolus peruvianus (Müll. Arg.) J.F. Macbr., Euphorbiaceae, the female designation, is used strictly as a female aphrodisiac. The use of any of these by the opposite sex is thought to have anti-aphrodisiac effect (Pachacuti-Yamqui, 1992).

A detailed description of preparation and use of C. crassa "Huanarpo macho" in an alcoholic beverage is given in (Bussmann et al., 2011a,b). In Peru this plant can be found in the regions of Cajamarca (San Ignacio, Chota and Jaén), Amazonas (Bagua), Lambayeque (Chiclayo), La Libertad (Trujillo), Pasco (Oxapampa) and Cusco (La Convención) (Brako and Zarucchi, 1993; Tropicos).

C. crassa is a hemi-parasitic plant, which grows on roots of other species. Likewise Balanophoraceae family consists of 17 genera and approximately 50 species (Tupac Otero et al., 2009). The natural distribution reaches from Costa Rica to Bolivia and the plant grows at altitudes from 1250 to 3600 m (Tupac Otero et al., 2009). C. crassa is not only an aphrodisiac plant there are also reports from ethanolic extracts which have shown biological activity against Staphylococcus aureus (Bussmann et al., 2010, 2011a,b). The ethanolic extract exhibit a toxicity of 116 μg/ml using a brine-shrimp lethality assay however, the water extract was found to be non-toxic (<10,000 μg/ml) for the same lethality assay (Bussmann et al., 2011a,b).

Currently, there is little information about constituents of C. crassa (Duff and Nickrent, 1997; Nickrent et al., 1997). A phytochemical investigation of the whole plants of C. crassa was thus performed as part of our work on the discovery of bioactive compounds from Peruvian herbal medicines. This led to isolation and characterization of β-sitosterol, lupenone, β-amyrone, lupeol, and β-amyrine.

In this work, the isolation and structure elucidation of these constituents are described.

Materials and methods

Used NMR, MS and HPLC machines and methods

NMR spectra (¹H, ¹³C, APT, H,H-COSY, HSQC, and HMBC) were recorded on a Varian Mercury 400 plus (400 MHz for ¹H, 100 MHz for ¹³C) and a Varian Mercury 300 plus (300 MHz for ¹H, 75 MHz for ¹³C) spectrometer, respectively, at 26 °C and the use of CDCl₃ as a solvent. The chemical shifts are reported relative to the residual solvent peak, which was used as an internal reference (¹H: 7.26 ppm, ¹³C: 77.16 ppm). Chemical shifts are given in ∆ values, coupling constants J in Hz; HR-MS spectra were performed with a Bruker Daltonics ESI-FT-ICR-MS APEX II [7 T] and HPLC Varian Pro star 360, vp 250/2 Nucleosil column 50-7.

Preparation of extracts

Isolation and structural elucidation of steroids

The tubers/roots of Corynaea crassa Hook. f., Balanophoraceae, were bought at the Mayorista Market in Trujillo and the Modelo Market in Chiclayo in Northern Peru. Dried and powdered roots (200 g) of C. crassa were extracted for 4 days at room temperature, using solvents of increasing polarity, namely, 1.5 L n-hexane and 1.5 I methanol. The solvent was then evaporated under reduced pressure, the n-hexane extract yielded 1.062 g and the methanol extract 4.240 g of crude product. Liebermann's reagent enabled us to determine remaining steroids and triterpenes in the methanolic extract, which was re-dissolved in water and extracted with dichloromethane yielding 906 mg. Both extracts were combined for a saponification process with 5% NaOH and we obtained in total 209 mg of steroids and triterpenes (TS1).

The plant material was taken from Bussmann and Sharon's collection (Voucher: Supporting information, p. 19).

Isolation by chromatography

TS1 (209 mg) was fractionated by column chromatography on silica gel eluting with n-hexane/dichloromethane gradient system (10:0.5 → 10:1 → 10:1.5 → 10:2 → 10.2.5 → 10:3) to give five fractions. Fraction 1 contained traces of two steroids (5.3 mg) seemed on the TLC plate and ¹H NMR but they were not possible to separate by CC and HPLC.

Fraction 2 (20.1 mg) was subjected to further HPLC with pure n-hexane (R_f: 0.81); After several purifications a lot of material was lost. Then the finally material was recrystallized from methanol/chloroform to yield β-amyrenone/lupenone (10.8 mg). Fractions 3 and 4 (62.9 mg) were further purified (Silica gel, n-hexane/DCM = 5:0.06 → 5:0.1 → 5:0.2 → 5:0.3 → 5:0.4) to yield 12.5 mg of β-sitosterol. And finally fraction 5 (18.8 mg) was purified by flash CC (n-hexane/DCM = 10:0.1 → 10:0.3 → 10:0.4 → 10.5 → 10:6 → 10:0.7) and HPLC (n-hexane/isopropanol = 9:0.0–9:0.02) then recrystallized from methanol/chloroform to yield 15.3 mg of β-amyrine/lupeol (R_f: 0.24). Finally, 2.8 mg of lupeol (1) was isolated by HPLC (n-hexane/isopropanol = 9:0.1).

Lupeol (1): White powder; MP. 215 °C; [α]_D = +26,4 (c = 0.4, CHCl₃); ¹H NMR (400 MHz, CDCl₃): ∆ (ppm) = 0.69 (1H, m, H-5), 0.77 (1H, s, H-24), 0.80 (1H, s, H-28), 0.84 (1H, s, H-25), 0.94 (1H, m, H-1b), 0.96 (1H, m, H-15b), 0.96 (1H, s, H-27), 0.97 (1H, s, H-23), 1.04 (1H, s, H-26), 1.10 (1H, m, H-12b), 1.19 (1H, m, H-22), 1.27 (1H, m, H-21), 1.29 (1H, m, H-11b), 1.30 (1H, m, H-9), 1.38 (1H, m, H-7), 1.39 (1H, m, H-6b), 1.39 (1H, m, H-18), 1.43 (1H, m, H-11a), 1.48 (1H, m, H-16), 1.52 (1H, m, H-6a), 1.61 (1H, m, H-15a), 1.61 (1H, m, H-2), 1.62 (1H, m, H-13), 1.64 (1H, m, H-1a), 1.70 (1H, m, H-12a), 1.70 (1H, s, H-30), 2.38 (1H, m, H-19), 3.18 (1H, dd, J = 11.0; 5.3 Hz, H-3), 4.56 (1H, s, H-29b), 4.68 (1H, s, H-29a).

¹³C NMR (100 MHz, CDCl₃): ∆ (ppm) = 14.8 (CH₃, C-27), 15.6 (CH₃, C-24), 16.1 (CH₃, C-26), 16.2 (CH₃, C-25), 18.1 (CH₃, C-28), 19.0 (CH₂, C-6), 19.8 (CH₃, C-30), 21.2 (CH₂, C-11), 25.3 (CH₂, C-12), 27.2 (CH₂, C-15), 27.5 (CH₂, C-2), 28.4 (CH₃, C-23), 30.1 (CH₂, C-21), 34.2 (CH₂, C-7), 35.9 (CH₂, C-16), 37.2 (C, C-10), 38.5 (CH, C-13), 38.7 (CH₂, C-1), 39.8 (C, C-4), 40.3 (CH₂, C-22), 41.1 (C, C-8), 42.8 (C, C-14), 43.2 (C, C-17), 47.8 (CH, C-19), 48.5 (CH, C-18), 50.9 (CH, C-9), 55.5 (CH, C-5), 79.3 (CH, C-3), 109.5 (CH₂, C-29), 151.2 (C, C-20); HR-MS: m/z 449.43 [M+Na]⁺, for formula C₃₀H₅₀O.

β-Sitosterol HR-MS: m/z 437.41 [M+Na]⁺, for formula C₂₉H₅₀O.

Lupenone/β-amyrone HR-MS: m/z 447.40 [M+Na]⁺, for formula C₃₀H₄₈O.

Lupeol/β-amyrine HR-MS: m/z 449.43 [M+Na]⁺, for formula C₃₀H₅₀O.

X-ray crystallography

Recrystallization of the lupenone/β-amyrone mixture from methanol/chloroform (1:0.3) gives colorless prisms. A crystal of the dimensions 0.3 mm × 0.15 mm × 0.15 mm was used for analysis. The intensities were measured on an IPDS1 diffractometer (Fa. STOE) and were corrected for Lorentz and polarization effects. The structure was solved by direct methods, and the refinement was performed with SHELX-97 (Sheldrick, 1997). Crystal data: a = 11.203 Å, b = 14.956(2) Å, c = 30.513(6) Å, V = 5112.5 ų, space group P2(1)2(1)2(1), Z = 8, D_calc = 1.103 mg/m³, λ = 0.71073 Å, μ (MoKα) = 0.064 mm⁻¹, F(000) = 1888, and T = 213(2) K, the total of 38,852 reflections (9556 independent, R_int = 0.082) in the range of 2.2° ≤ θ ≤ 26.1° and 559 refined parameters. The final parameters were R₁ = 0.0442, R₂ = 0.0948 for 3195 observed reflections with I > 2σ (I) and a goodness-of-fit = 0.558.

Crystallographic data for the structure determined in this paper have been deposited at Cambridge Crystallographic Data center (CCDC reference number is CCDC 1008248) and can be obtained free of charge from Cambridge Crystallographic Data Centre CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: [phone]; [e-mail].

Results and discussion

Spot tests were used for the qualitative determination of secondary metabolites present in the crude extract of C. crassa, as it was described in (Dominguez, 1973; Harborne, 1984). We have identified steroids, triterpenes, flavonoids, cardiotonics, tannins and anthocyanines, whereas alkaloids have not been detected. According to Liebermann–Burchard's tests, the experiments also have shown an intense green dark coloration for steroids and triterpenes, indicating that these secondary metabolites are in the crude extract.

The steroid β-sitosterol, was one of the main products isolated from the n-hexane extract and purification by chromatography with n-hexane/dichloromethane. β-Sitosterol itself is widely distributed in the plant kingdom and used in herbal therapy, especially for benign prostatic hyperplasia (BPH) (Berges et al., 1995). The structure was confirmed by NMR, HR-MS and comparison with the literature results (Wright et al., 1978; Faizi et al., 2001).

In a next step, the n-hexane extract furnished a 1:1 mixture of the pentacyclic isomeric triterpenes lupenone and β-amyrone. It was not possible to separate both compounds using chromatographic methods. Finally, we were successful to identify the structure of the complex mixture by use of 1D and 2D NMR spectroscopy, X-ray and HR-MS. Our results are in agreement with the literature data (Wenkert et al., 1978; Carpenter et al., 1980). These isomeric triterpene ketone derivatives are only distinguished in its structure by their 5- and 6-membered E ring, respectively, and give the same high resolution mass spectrum. In the ¹H NMR spectrum of the mixture we observed three signals of olefinic protons in a ratio 1:1:1. The signal at 5.18 ppm was assigned to the methine proton in β-amyrone (Fig. 1), while the signals at 4.66 and 4.54 ppm, respectively, result from the methylene protons of lupenone.

Fig. 1
Characteristical part of the ¹³C NMR spectrum of the 1:1 lupenone/β-amyrone mixture.

The ¹³C NMR spectrum shows typical signals at 218.03 and 217.65 ppm corresponding to the carbonyl groups. The olefinic carbon atoms appear at 109.51 (=CH₂) and 150.80 ppm (=C-) (lupenone), and at 121.59 (=CH-) and 145.29 ppm (=C-) (β-amyrone) (Fig. 1).

We were able to recrystallize the triterpene mixture using methanol/chloroform (1:0.2) to give crystals enough suitable for X-ray analysis. The crystal structure unambiguously confirms the interpretation of the spectra and the structure of the isolated products. In the orthorhombic unit cell there are two structural constitution isomers of the formula C₃₀H₄₈O: lupenone and β-amyrone (Fig. 3).

Fig. 2
Characteristical part of the ¹³C NMR spectrum of the 1:1 lupeol/β-amyrine mixture.

Fig. 3
Molecular structures of lupenone (A) and β-amyrone (B) (ellipsoids with 50% probability).

The formation of a 1:1 mixed crystal is due to the close resemblance of the conformation with the rings A, B, C and D. A calculation of the puckering parameters according to (Cremer and Pople, 1975) gives parameters which agree with a C (chair) – conformation. Only the C-ring of β-amyrone has an E (envelope) – configuration. The reason for this difference is the double bond between C12B–C13B (1.335 (5) Å). A fitting of both molecular structures shows the broad consistency between the rings A, B, C and D (Fig. 4).

Fig. 4
Fitting of the molecular structures of lupenone and β-amyrone. In the crystal structure the two different molecules also form separate helical arrangements along the b-axis.

Our X-ray data are in agreement with the X-ray structures of lupenone and β-amyrone obtained as separated compounds by isolation from other plants (Dampawan et al., 1977; Yan et al., 1989; Jaiswal et al., 2004).

Besides the lupenone/β-amyrone mixture a combination of lupeol/β-amyrine (also in 1:1 ratio; Fig. 2), could be isolated in lower yield (30% compared to lupenone/β-amyrone). The structure of these triterpenes was also confirmed by 1D and 2D NMR techniques, MS measurements and comparison with the literature results (Wenkert et al., 1978; Carpenter et al., 1980). In the ¹H NMR spectrum the olefinic protons appear at 5.18 (=CH- of β-amyrine), 4.68 and 4.56 (=CH₂ of lupeol), and additionally the typical CH-O absorptions at 3.20 ppm. In the ¹³C spectrum the olefinic carbons appear nearly unchanged (151.03, 145.29, 121.85 and 109.45 ppm) in comparison to the spectrum of the lupenone/β-amyrone mixture. But instead of carbonyl signals now two closely related CH-O signals appear at 79.12 and 79.10 ppm.

After additional HPLC purification of this mixture it was possible to isolate lupeol as nearly pure compound for separate NMR characterization.

Interestingly, the common appearance of the combinations lupenone/β-amyrone and lupeol/β-amyrine was observed for first time. In contrast, the combination of lupanone/lupeol already was found in Tamarindus indica L., Fabaceae (Mathur, 2012), and the combination of β-amyrone/β-amyrine was isolated and characterized from Diospyros morrisiana Hance, Ebenaceae (Yan et al., 1989).

The formation of both lupenone/β-amyrone and lupeol/β-amyrine each in a 1:1 ratio was unexpected because lupenone and lupeol were formed via the same lupenyl cation, whereas β-amyrine and β-amyrone were formed via the oleanyl cation in the proposed biosynthetical pathway (Gallo and Sarachine, 2009; Seo et al., 1981).

A complex mixture of all these 4 pentacyclic triterpenes from the same plant has never been observed before. Otherwise, the individual compounds have been isolated and described as constituents in different aphrodisiac plants (Mathur, 2012; Mazumder et al., 1999; Corrêa et al., 2009; Imman et al., 2007).

Conclusion

The present paper describes the isolation and characterization of five constituents from C. crassa: β-sitosterol and two interesting 1:1 mixtures of triterpenes: lupenone/β-amyrone and lupeol/β-amyrine. The results may be helpful in further investigations of the biological activity of these natural compounds.

Acknowledgments

The authors are grateful to Mrs. Ramona Oehme (University of Leipzig, Institute of Analytical Chemistry, Germany) for the measurement of the MS spectra.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bjp.2015.02.007.

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Publication Dates

• Publication in this collection
  Mar-Apr 2015

History

• Received
  9 Sept 2014
• Accepted
  24 Feb 2015