Next Article in Journal
Enrichment of Polyglucosylated Isoflavones from Soybean Isoflavone Aglycones Using Optimized Amylosucrase Transglycosylation
Previous Article in Journal
Production and Characterization of Bioplastic by Polyhydroxybutyrate Accumulating Erythrobacter aquimaris Isolated from Mangrove Rhizosphere
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 25
Issue 1
10.3390/molecules25010180
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Iridoids and Amino Acid Derivatives from the Paraguayan Crude Drug Adenocalymma marginatum (ysypó hû)

by
Guillermo Schmeda-Hirschmann
1,*,
Alberto Burgos-Edwards
1,
Felipe Jiménez-Aspee
2,
Daniel Mieres-Castro
1,
Cristina Theoduloz
3,
Lisa Pormetter
1,
Ramon Fogel
4,
Claudia Céspedes
4,
Nelida Soria
4 and
Sintya Valdez
4
1
Laboratorio de Química de Productos Naturales, Instituto de Química de Recursos Naturales, Universidad de Talca, Campus Lircay, Talca 3460000, Chile
2
Departamento de Ciencias Básicas Biomédicas, Facultad de Ciencias de la Salud, Universidad de Talca, Campus Lircay, Talca 3460000, Chile
3
Laboratorio de Cultivo Celular, Facultad de Ciencias de la Salud, Universidad de Talca, Campus Lircay, Talca 3460000, Chile
4
Centro de Estudios Rurales Interdisciplinarios, Oliva 1019, Edificio Lider V, Piso 17, oficina 172, Asunción 01421, Paraguay
*
Author to whom correspondence should be addressed.
Molecules 2020, 25(1), 180; https://doi.org/10.3390/molecules25010180
Submission received: 5 December 2019 / Revised: 19 December 2019 / Accepted: 27 December 2019 / Published: 1 January 2020
(This article belongs to the Section Natural Products Chemistry)
Browse Figures
Review Reports Versions Notes

Abstract

:
The crude drug ysypó hû (Adenocalymma marginatum DC., Bignoniaceae) is used traditionally by the Guarani of Eastern Paraguayan as a male sexual enhancer. The aim of the present study was to identify the main constituents of the crude drug and to evaluate the in vitro inhibitory activity towards the enzyme phosphodiesterase-5 (PDE-5). The main compounds were isolated by counter-current chromatography (CCC). The metabolites were identified by spectroscopic and spectrometric means. The chemical profiling of the extracts was assessed by high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS/MS). The crude extract and main isolated compounds were tested for their PDE-5 inhibitory activity using commercial kits. The iridoid theviridoside and 4-hydroxy-1-methylproline were isolated as the main constituent of the crude drug. Four chlortheviridoside hexoside derivatives were detected for the first time as natural products. Chemical profiling by HPLC-MS/MS led to the tentative identification of nine iridoids, six phenolics, and five amino acids. The crude extracts and main compounds were inactive towards PDE-5 at concentrations up to 500 µg/mL. Iridoids and amino acid derivatives were the main compounds occurring in the Paraguayan crude drug. The potential of ysypó hû as a male sexual enhancer cannot be discarded, since other mechanisms may be involved.

Graphical Abstract

1. Introduction

The Guarani Amerindians inhabited large parts of eastern South America before the European conquest. The group included the Tupi and Guarani people, living from the costs of the Atlantic Ocean to the subtropical forests of Paraguay and eastern Argentina. The Tupi–Guarani Amerindians are closely related, sharing a common language and playing a relevant role in the South American traditional medicine and culture [1]. Paraguayan traditional medicine has strong roots in Guarani culture and has provided the world with relevant food and medicinal plants, including the tea mate (Ilex paraguariensis) [2], the sweet herb stevia (“kaa hee”, Stevia rebaudiana) [3], and hundreds of medicinal plants widely used in South America.
The use of medicinal plants by the Guarani people was described by Pedro de Montenegro (1770), during the Spanish Jesuit Missions in Paraguay [4]. Plant and animal drugs were selected based on empirical knowledge and the self-experience of the healers. The use of sexual enhancers by the Guarani people is not common. However, fertility control was widespread, with plants used as contraceptive and as abortifacients [5]. The Bignoniaceae Adenocalymma marginatum (Cham.) DC. (Figure 1) is commonly known as “ysypó hû” (black vine) by the Pai Tavytera and Mbyá Amerindians, who live in northeastern Paraguay. The plant has the synonyms Adenocalymma marginatum var. marginatum, A. marginatum var. polystachyum DC, and the basionym Bignonia marginata Cham. [6]. The plant, fresh or in decoction, is recommended to improve fecundity and as a male sexual enhancer [7,8]. The crude drug includes the aerial parts of the plant (leaves and stem) but consists mainly of stems. The root was included in this study for comparison purposes. For the preparation of the drug, the stem and leaves are powdered, and a handful is placed in 1 L water and boiled for 3–5 min. After cooling, the decoction is taken orally (about 200 mL) two times a day, as long as necessary. The preparation is also included in mate tea, where mate is the carrier of the medicinal plants [2]. It is also used to relieve rheumatism and as anti-diarrheic [7,8].
The use of plants and animal parts as sexual enhancers is a known practice worldwide [9]. Since ancient times, several natural products have been used as sexual enhancers for the treatment of erectile dysfunction, including yohimbine from Pausinystalia yohimbe (Rubiaceae), forskolin from the Indian herb Plectranthus barbatus (Lamiaceae), or the “Peruvian Viagra” Lepidium meyenii (Brassicaceae), among many others [10]. They are popularly considered safe and “natural”, and no strict regulation is applied to control their sale. Unfortunately, a study published in 2015 showed that 61% of a total of 150 supplements were altered with synthetic phosphodiesterase-5 (PDE-5) inhibitors, including the commercial drug sildenalfil, or structurally modified analogs [11]. We recently reported the chemical composition and potential male sexual enhancer activity of Campsiandra angustifolia, Minquartia guianensis, Swartzia polyphylla, and Tynanthus panurensis, as well as from two traditional preparations of the Peruvian Amazon. In our experiment, C. angustifolia was found to be a potent inhibitor of PDE-5 in vitro [12].
The occurrence of the iridoids ipolamiide, strictoloside, and theviridoside in leaves of A. marginatum from Rio Grande do Sul (southern Brazil) has been reported [13]. However, no information is available on the chemical composition of the Paraguayan crude drug, nor on the male sexual enhancement capacity of A. marginatum. Hence, the aims of the present study were to isolate and characterize the main secondary metabolites from the Paraguayan crude drug ysypó hû. Additionally, the potential male sexual enhancer capacity was evaluated by the in vitro inhibitory capacity of this traditional preparation towards the enzyme phosphodiesterase-5.

2. Results and Discussion

2.1. CCC Isolation of the Main Compounds from Ysypó Hû

The TLC analysis of the crude drug extracts (Table 1 and Figure 2) showed that the main compounds were UV inactive and were visualized only after spraying with p-anisaldehyde/sulfuric acid and heating. The semi-purified fractions 8–15 from the Sephadex of the stem extract were worked up to obtain the main compounds using counter-current chromatography (CCC). The solvent system selected for the CCC was made of TBME/BuOH/ACN/H2O 1:3:1:5 (v/v/v/v), supplemented with 0.1% trifluoroacetic acid. As the main compounds from the crude drug were UV inactive, partition ratios (KD) values were calculated by visualizing the compounds after spraying with p-anisaldehyde–sulfuric acid and scanning the TLC plates for densitometric analysis. The KD values of the different solvent systems and a figure of the original TLC used are shown in Table 1. The theoretical KD value for the main spot in the selected solvent system was 0.21. The system was selected due to its good stability, short settling time in the presence of the sample, and the low KD value, which allowed the separation of the compound with a low consumption of solvents. After setting the CCC in the selected conditions, 70.3% retention of the stationary phase was observed. The retention volume (VR) was calculated following the equation of Ito [14], which gave a VR of 222 mL for the main peak. After the sample was injected, the detection of the main compound in TLC analysis began at Tube 62 (Figure 2). Considering that the pump was set at a flow rate of 4 mL/min, and the recollection was 1 min/tube, the experimental KD value of the main compound was 0.28, in agreement with the theoretical calculation. Fractions from the CCC separation were pooled according to similarities of the TLC profiles.
The main compound found in fractions 62–71 (103 mg) of the CCC was a colorless solid, soluble in methanol and insoluble in chloroform. The 1H-NMR spectrum of the compound showed two singlets at δ 7.52 and δ 5.72 ppm, suggesting a conjugated double bond and a trisubstituted double bond. In addition, a doublet at δ 5.55 and a second doublet at δ 4.65 agreed with a hemiacetal and an anomeric H, respectively. Two geminal coupling doublets at δ 4.25 and δ 4.14 ppm indicated a primary alcohol, and the singlet at δ 3.75 agreed with a methyl ester, supporting an iridoid skeleton.
The 13C-NMR spectrum showed 17 C signals, including 2 trisubstituted double bonds, a carboxylic acid methyl ester at δ 166.96 and 50.41 ppm, and 6 C signals assignable to a hexose. The structure was established following the heteronuclear multiple bond correlation (HMBC) experiments. The mass spectrum of the sodium adduct (calculated 427.1216, found 427.1196) indicated a molecular formula of C17H24O11, in full agreement with the iridoid glucoside theviridoside [15,16]. The acetylated fraction pool 8–15 afforded the disaccharide sucrose (as octaacetate) and theviridoside pentaacetate after purification by column chromatography (Figure 3).
Theviridoside. Molecular formula: calculated for C17H24O11: 404.1319; found: 404.1325; theviridoside pentaacetate: 614.1861 (calculated for C27H34O16: 614.1847). The NMR data of theviridoside and its pentaacetate are summarized in Table 2.
The fractions 58–60 (50 mg) from the CCC separation contained a compound showing in the FT-IR spectrum absorption bands of a carboxylic acid function at 1682 cm−1 and hydroxyl at 3369 cm−1. The 1H-NMR spectrum presented a methyl singlet at δ 3.08, compatible with a N-methyl group, a dd at δ 4.36 (1H), a m at δ 4.55 (1H), and two pairs of geminal coupling H forming a CH-CH2-CH-CH2 sequence. The 13C-NMR spectrum indicated six C, one of them for the carboxylic acid at δ 170.38, a N-methyl group at δ 42.79, two triplets for the CH2 groups, and two doublets, assigned by correlation spectra as the -CHOH and α-amino acid CH from a small heterocyclic ring. The compound was identified as 4-hydroxy-1-methyl proline (compound I), in agreement with the molecular formula C6H11NO3 calculated from the positive ion mass spectrum. The structure of the compound is shown in Figure 3 and the NMR data are summarized in Table 3. The compound has been previously reported as a stress response metabolite from Tamarix species [17] and from the toxic plant Ipomoea carnea [18]. Fractions 65–70 (24 mg) afforded a mixture of monosaccharides and the amino acids isoleucine and proline.
4-hydroxy-1-methyl proline. Molecular formula: C6H11NO3. Calculated for C6H11NO3: 145.0739, found (QTOF, positive ion mode): 146.0584. 146.0584 (70), 100.0665 (100), 82.0639 (32) C5H9NO: 99.0684; C5H7N: 81.0578 FT-IR (KBr, film): 3369, 3328, 1682, 1627, 1401, 1346, 1196.

2.2. Chemical Profiling of the Crude Drug

Little is known of the chemistry of Adenocalymma species. C-4 carboxyl iridoids were identified in the leaves of a Brazilian sample of A. marginatum [13]. In the Paraguayan crude drug, CCC isolation and HPLC-MS analyses allowed the identification of iridoids, phenolics, and amino acids, most of which were reported for the Brazilian sample. The HPLC-MS chromatogram is presented in Figure 4. The identification of the constituents and the occurrence in stem and root (plant parts used in Guarani medicine) is shown in Table 4.

2.2.1. Iridoid Glycosides

The main compound of the Paraguayan crude drug was isolated and fully identified as theviridoside (8) (Table 2, Figure 3). Theviridoside presents a [M − H]− ion at m/z 403 amu (atomic mass units), and shows a characteristic fragment at m/z 241 due to the neutral loss of hexose. This fragmentation pathway has been described previously [19]. Theviridoside was previously reported in the roots of the Apocynaceae Thevetia peruviana [15,16]. Related compounds have been previously identified in the Rubiaceae Aitchisonia rosea [20] and Genipa americana [21]. Minor constituents were identified or tentatively identified by examination of the fragmentation patterns in MSn, as well as by comparison with literature [22]. Compounds 2, 5, and 10 showed the iridoid base peak at m/z 241, differing in the identity and placement of the substituents. Compounds 2 and 5 showed a common base peak ion at m/z 727 amu, with the consecutive neutral loss of three hexoses being tentatively identified as theviridoside dihexosides. Compound 10 showed a base peak ion at m/z 565, with a neutral loss of 324 amu, leading to the fragment at m/z 241 amu. The compound was identified as theviridoside hexoside. The compounds 3, 4, 6, and 9 showed the loss of 36 amu, leading to the base ion at m/z 727 amu for compound 3 and m/z 565 amu for compounds 4, 6 and 9, respectively. The tandem mass spectrometry (MS2) fragmentation showed the same pattern as theviridoside dihexoside or hexoside, suggesting derivatives of theviridoside. The [M − H]− ions of compounds 3, 4, 6, and 9 showed two peaks separated by two atomic mass units in a 3:1 ratio, in agreement with the isotopic peaks of 35Cl (75.8%) and 37Cl (24.2%), supporting the presence of a chlorine atom in the compounds.
Compounds 4, 6, and 9 were tentatively assigned as chlortheviridoside hexoside derivatives, while compound 3 was assigned as a chlortheviridoside dihexoside derivative. The different Rts suggested either differences in the identity of the hexoses and/or placement of the hexose in the iridoid core. The placement of the chlorine atom in the compounds remains to be established. In iridoids, it is common to find the chlorine atom placed at C-7 [23]. Examples include mentzefoliol and glucosylmentzefoliol from Mentzelia cordifolia (Loasaceae), stegioside I from the Lamiaceae Physostegia virginiana ssp. virginiana (Lamiaceae), and 6-O-p-hydroxybenzoylaystasioside and glutinoside derivatives from Catalpa fructus (Bignoniaceae), as well as in Asystasiosides from Premna subscandens (Verbenaceae) [23]. To the best of our knowledge, compounds 3, 4, 6, and 9 have not been reported previously as natural products.
Compound 7 presented a molecular ion at m/z 659 amu, and the neutral loss of 256 amu and a hexose (162 amu), leading to the MS2 ions at m/z 402 and 241 amu, in agreement with theviridoside. The first neutral loss exceeded by 14 amu the molecular weight of theviridoside aglycon (242 amu). Thus, compound 7 was assigned as a methyl theviridoside aglycon–theviridoside dimer. Related compounds have been described from Saprosma scortchenii (Rubiaceae) [24,25].
Related compounds, including iridoid glycoside polymers, have been described from Eucommia ulmoides and showed antioxidant activity in vitro [26]. Dimeric iridoid glycosides linked to a sugar and/or phenyl moiety have been described for Globularia trichosantha [27]. Iridoids have been reported from Bignoniaceae species including Campsis chinensis [28] and Pithechoctenium crucigerum [29].

2.2.2. Phenylpropanoid Glycosides

Compound 1 presented a molecular ion at [M − H]− 341 amu, with the neutral loss of an hexose (−162 amu), leading to a MS2 ion at m/z 179 amu, in agreement with caffeic acid. The compound was tentatively identified as caffeoyl hexoside. Compound 11 presented a [M − H]− ion at m/z 625, fragments to a base peak at m/z 461 amu, and a secondary ion at m/z 315 amu. The compound is related to acteoside/isoacteoside, differing in the dihydrocaffeoyl unit linked to the glucose core. Compound 12 presented a [M − H]− ion at m/z 769 amu and the neutral loss of 162 amu, leading to the base peak at m/z 607. The further loss of a rhamnose and the ion at m/z 461 is in agreement with angoroside B or its isomer. Angoroside B has been reported as a constituent of Scrophularia scopolii, Populus trichocarpa, and P. deltoides with antibacterial activity [30]. Compounds 13 and 14 showed the neutral loss of a caffeoyl (−162 amu) and a rhamnose (−146 amu), leading to the fragment ions at m/z 461 and 315 amu, in agreement with acteoside (verbascoside) isomers. According to References [31,32], acteoside elutes first, followed by isoacteoside. Both compounds differ in the relative placement of the caffeoyl moiety in the glucose core, being at 4-O for acteoside and 6-O for isoacteoside. The caffeoyl–phenethyl glycosides acteoside and isoacteosides have been described as antineoplastic agents [33]. Compound 15 presented a [M − H]− ion at m/z 515 and fragment ions at m/z 353 and 173, in agreement with dicaffeoylquinic acid [34]. The exact placement of the caffeic acid moieties in the compound remains to be established.

2.2.3. Amino Acids and Derivatives

HPLC-MS/MSn analysis of the extract in the positive ion mode allowed the identification of nitrogen-containing compounds. The occurrence of compound I was confirmed by the MS analysis, in full agreement with the literature [17,18,35]. The compound has been previously reported in Sideroxylon obtusifolium (Sapoteaceae), a medicinal species from Northeast Brazil with anti-inflammatory [36] and wound healing properties [37]. The authors reported that the compound at doses ranging from 25–100 mg/kg decreased licking time and paw edema in the formalin test, as well as a complete reversion of the increased number of polymorphonuclear cells in the inflammation point. Additionally, the compound decreased the expression of iNOS, TNF-α, COX-2, and NF-κβ [36]. Compounds II and III presented [M + H]+ ions at m/z 99.6 and 81.5 amu, respectively. Compound II showed the neutral loss of water (−18 amu), leading to the base peak at m/z 81.5 amu, supporting its close relationship with compound III. They were assigned as 1-methyl-2,3-dihydro-3-hydroxypyrrol and 1-methylpyrrol, respectively. The compounds IV presented a molecular ion at [M + H]+ m/z 115.7 amu. The neutral loss of 46 amu (H2O + CO) led to the MS2 ion at m/z 70 amu. The fragmentation pattern was in agreement with the cyclic amino acid proline [38]. Compound V presented a molecular ion at [M + H]+ m/z 132.1 amu. The neutral loss of 46 amu (H2O + CO) led to the MS2 ion at m/z 86.1 amu. The consecutive loss of −17 amu (NH3) was in full agreement with the branched amino acid isoleucine [38]. The HPLC traces are depicted in Figure 4.
The chemistry of the Paraguayan A. marginatum differs from that of Adenocalymma species previously investigated by the absence of sulfur-containing compounds and the occurrence of iridoids, phenylpropanoid glycosides, and amino acid derivatives in ysypó hû. Bioactivity studies on Adenocalymma have included a screening of the antimicrobial effect of climbers from the Bignoniaceae family [39] and the influence of aqueous and ethanol extracts of Pseudocalymma alliaceum on hematological parameters in rats [40].

2.3. Effect of the Extracts and Main Constituents on Phosphodiesterase-5 (PDE-5) Activity

The Paraguayan crude drug ysypó hû is traditionally used as a male sexual enhancer by the Guarani people. The root of the Bignoniaceae Anemopaegma arvense (Vell.) Stellfeld & J.F. Souza (catuaba) is traditionally used as an aphrodisiac in central Brazil [41]. PDE-5 is the predominant cGMP-metabolizing enzyme that is expressed in the cavernosal tissue and in penile arteries, and it is the pharmacological target of sildenafil (Viagra®), vardenafil (Levitra®), and tadalafil (Cialis®). These three drugs are clinically used for the treatment of erectile dysfunction because they are potent and selective PDE-5 inhibitors, which results in increased levels of cGMP in the smooth muscle cells of the penile corpus cavernosum, a decrease in the intracellular calcium, and desensitization of proteins to the effects of calcium, producing smooth muscle relaxation and penile erection [42]. The ysypó hû extracts and main compounds were assessed as potential PDE-5 inhibitors. Under our experimental conditions, neither the crude root, stem extracts, nor the main compounds presented inhibitory activity at concentrations up to 500 µg/mL. Under the same experimental conditions, sildenafil citrate showed an IC50 of 21.07 ± 0.61 µg/mL. Despite the lack of inhibition of PDE-5 by ysypó hû extracts and the main constituents, other mechanisms for sexual enhancement cannot be ruled out. Several natural products have demonstrated effectiveness against erectile dysfunction [43]. The indole alkaloid yohimbine from Corynanthe yohimbe acts as an antagonist on α2-adrenergic receptors, increasing sympathetic outflow to the periphery, increasing vasomotor activity, and creating an aphrodisiac sensation [44]. The iridoid glycosides morroniside and loganin, from the traditional chinese medicine Xiao Ke Zheng, protected against diabetes-mellitus-induced testicular damage in rats by increasing sex hormone levels (GnRH, LH, and FSH) and testicular/body weight ratio, as well as decreasing oxidative stress and cellular apoptosis in the testis [45]. The aphrodisiac properties of the Andean root of Lepidium meyenii (maca) have been widely described in literature [46,47]. In humans, the extract significantly improved sexual desire compared to placebo, independently of serum testosterone levels [48]. Despite all the evidence, the mechanism of action of L. meyenii is not clear so far. Thus, despite the lack of inhibition presented by A. marginatum towards PDE-5, further investigations are needed in order to clarify its potential as a male sexual enhancer.

3. Materials and Methods

3.1. Plant Material

The stems and roots of Adenocalymma marginatum DC. were collected on December of 2015 at Mondaymi, Distrito de San Joaquin, Departamento de Caaguazú, Paraguay (25°9’21,39”S, 56°4’44,16”W). A voucher herbarium specimen (Céspedes 1628) was deposited at the Herbario FCQ, Facultad de Ciencias Químicas, Universidad Nacional de Asunción, Asunción, Paraguay. The air dried plant material was powdered and extracted three times with EtOH:H2O (70:30, v/v). The combined extracts were evaporated under reduced pressure and lyophilized to yield the crude extract. The w/w extraction yields were 18% and 23% for the stem and roots, respectively. The EtOH:H2O (70:30) extract was preferred over the infusion for the higher extraction yields.

3.2. Isolation of the Main Compounds

The lyophilized extracts of stems and roots were worked with separately in order to obtain fractions and to identify the constituents of the crude drug. To remove fats and chlorophylls, the root and stem extract were partitioned against CHCl3. The root extract (53.9 g) yielded a CHCl3-soluble (1.5% w/w yield) and a CHCl3-insoluble fraction (98.5% w/w). The stem extract (95.56 g) yielded a CHCl3-soluble (0.95% w/w) and a CHCl3-insoluble (99.05% w/w) fraction. The root and stem CHCl3-insoluble extracts, containing the polar constituents, were compared by TLC (silica gel) using different solvent mixtures as mobile phase. Both plant parts presented similar profiles (data not shown).
A total 5.5 g of the polar, CHCl3-insoluble stem extract was loaded into a Sephadex LH-20 column (column length: 105 cm; internal diameter: 5.2 cm; Sephadex load: 45 cm, Sigma-Aldrich, St. Louis, MO, USA) previously equilibrated with MeOH. The procedure was carried out two times in order to separate a total of 11 g of the stem extract. In both cases, the void volume was 200 mL. Thirty fractions of 60 mL each were collected. Fractions were pooled according to the TLC analysis (Silica gel; EtOAc: formic acid (98–100%): H2O:acetone 10:2:3:1) as follows: Fractions 1–6 did not contain compounds of interest and were discarded; Fraction 7 (225 mg) contained a main compound with Rf 0.08; Fractions 8–15 (9.16 g) showed in TLC two compounds with Rf. 0.08 and 0.27; Fractions 16–18 (320 mg) contained a main compound with Rf 0.27. Both compounds were revealed as blue spots after spraying the TLC plate with anisaldehyde and heating. Fractions 19–30 did not contain compounds of interest and were discarded. Fractions 8–15 were pooled and further purified by counter-current chromatography, as described in the following paragraphs. A total 1 g of the pooled Fractions 8–15 was acetylated (pyridine/acetic anhydride, 36 h, room temperature) and the major compound was purified by column chromatography (68 cm length, ID: 3.2 cm) on silica gel (88 g), using a PE:EtOAc 1:1 v/v mixture as eluent (isocratic). Some 170 fractions of 10 mL each were collected and combined in different pools according to the chromatographic behavior on TLC (silica gel, PE:EtOAc 1:1 v/v, detection: anisaldehyde/sulfuric acid and heating).

3.3. Counter-Current Chromatography (CCC) Isolation of Main Compounds

The CCC separation of the main compounds from Sephadex Fractions 8–15 was performed on a J-type Quattro MK5 Lab Prep equipment (AECS, Wales, UK). The CCC is equipped with four PTFE coils of 5 m × 2.16 mm (ID), with a total column volume of 500 mL and 120 mm of revolution radius. A manual sample injection valve with a 10 mL loop was used to introduce the sample into the column. The injection volume was 10 mL. The choice of the proper biphasic system for the CCC separation was carried out according to Reference [14]. Several biphasic solvent systems were tested to obtain suitable partition ratios (KD). The solvent systems were based on different combinations of tert-butylmethylether (TBME), 1-butanol, acetonitrile (ACN), and water acidified with 0.1% of trifluoroacetic acid (TFA) (TBME/BuOH/ACN/H2O) in different ratios. For the evaluation, approximately 5 mg of the sample was dissolved in a test tube containing 4 mL of the thoroughly equilibrated solvent systems to be tested (1:1, v/v). The test tubes were shaken, and the compounds were allowed to partition between the two phases. After the equilibration, the phases were separated and analysed by TLC in ALUGRAM® plates (Macherey-Nagel GmbH & Co, Düren, Germany), using as mobile phase EtOAc:formic acid:H2O:acetone (10:2:3:1 v/v/v/v), and revealed with p-anisaldehyde-sulfuric acid. The KD value of the main compound was calculated using a densitometric analysis of the TLC spots in the superior and inferior phases using the Plot Spot function of ImageJ software (ver. 1.50i, NIH, Bethesda, MD, USA). Briefly, TLC plates were photographed and the areas-under-the-curve (AUCs) of the pixels observed in the TLC spots were determined. The KD values were calculated, dividing the AUC from the organic phase vs. the AUC from the aqueous phase, in order to carry out separations in the head-to-tail mode. The KD values are summarized in Table 1. The day of the experiment, 2 L of the selected biphasic solvent system were prepared and separated in a separatory funnel, and then degassed by ultra-sonication for 15 min. The system was filled with the upper stationary phase using a HPLC pump (Serie II, Scientific Systems Inc., State College, PA, USA). The lower aqueous mobile phase was pumped at a flow rate of 4.0 mL/min. Rotation speed was then set at 680 rpm and temperature was maintained at 25 °C during all separations. After reaching hydrodynamic equilibrium, the sample was dissolved in 10 mL of a 1:1 v/v mixture of the upper and lower phase, sonicated, filtrated through a Clarinert Syringe filter 0.45 μm, 25 mm (Agela Technologies, Torrance, CA, USA), and manually injected into the CCC system. The injection volume was 10 mL. Fractions were collected using a Gilson FC 203B (company, Middleton, WI, USA) set at 0.8 min/tube and pooled after TLC comparison with the system described above. A total of 80 fractions were collected. To end the CCC, rotation was stopped, and the column content was extruded with MeOH at 6 mL/min. The separation by CCC was carried out two times, with a total mass injection of 1 g.

3.4. HPLC-DAD-MS/MSn Analysis

The extracts were compared by HPLC-DAD to obtain the profiles of the plant stem and leaves and to set-up the conditions for HPLC-MS/MS analysis. The hydroalcoholic extracts from stem and roots of A. marginata were analyzed using a Shimadzu Prominence chromatographer (Shimadzu Corporation, Kyoto, Japan). The HPLC was equipped with a LC-20AT pump, CTO-20AC column oven, and SPD-M20A UV diode array detector, operated by Labsolution software (ver. 5.6, Shimadzu Corp., Kyoto, Japan)). A MultoHigh 100 RP 18-5 µm (250 × 4.6 mm) column (Cs-Chromatographie Service GmbH, Langerwehe, Germany) was used. The column oven was set at 25 °C. Approximately 10 mg of each extract was dissolved in 1 mL MeOH, filtered through a 0.45 µm PTFE filter, and injected. The gradient solvent system consisted of H2O and formic acid (99:1, v/v, solvent A) and acetonitrile (ACN, solvent B). The initial composition was 10% B. The gradient was started with 90% A and 10% of B. The solvent ratio was then changed as follows: at min 15, 85% A and 15% B; at min 20, 85% A and 15% B; at min 25, 82% A and 18% B; at min 50, 82% A and 18% B; at min 80, 70% A and 30% B. The solvent ratio was then returned to the initial conditions and the column was stabilized for an additional 10 min before the next injection. The flow rate was 0.5 mL/min and the injected volume was 20 μL. The compounds were monitored at 280 nm.
Mass spectra were recorded using an Agilent 1100 (Agilent Technologies Inc., Santa Clara, CA, USA) liquid chromatography system connected through a split to an Esquire 4000 Ion Trap LC/MSn system (Bruker Daltonics, Bremen, Germany). Ionization was performed at 3000 V, assisted by nitrogen as nebulizing gas at 50 psi and as drying gas at 365 °C with a flow rate of 10 L/min. Negative ions were detected using full scan (m/z 20–2200) and normal resolution (scan speed 10,300 m/z/s; peak with 0.6 FWHM/m/z). The trap parameters were set in the ion charge control (ICC) using manufacturer default parameters, and a maximum accumulation time of 200 ms. The mass spectrometric conditions for the positive ion mode were as follows: electrospray needle, −2500 V; skimmer 1, 19.2 V; skimmer 2, 5.7 V; capillary exit offset 1, 33.0 V; capillary exit offset 2, 52.1 V. The mass spectrometric conditions for the negative ion mode were as follows: electrospray needle, 3500 V; skimmer 1, 20.3 V; skimmer 2, 6.0 V; capillary exit offset 1, 68.2 V; capillary exit offset 2, 88.5 V. The scan mode was performed at a speed of 13,000 m/z/s, in the range of 50 to 1200 m/z. Collision-induced dissociation (CID) spectra were obtained with a fragmentation amplitude of 1.00 V (MS/MS) using helium as the collision gas, and were automatically controlled through SmartFrag option.

3.5. Inhibition of Phosphodiesterase (PDE-5)

The inhibition of PDE-5 was carried out following the manufacturer’s instructions of the kit (ab139460, Abcam, Cambdrige, UK), as described in Reference [12]. Crude extracts from stem and roots, as well as the main isolated compounds, were evaluated in concentrations ranging from 0–500 μg/mL. Sildenafil citrate (Europharma, Sao Paulo, Brazil) was used as the positive control for inhibition of PDE-5. Absorbance was read at 600 nm in a microplate reader (ELx800, Biotek, Winooski, VT, USA). The results are expressed as the concentration of sample that inhibited PDE-5 by 50% (IC50, μg/mL).

4. Conclusions

Chemical profiling of the Paraguayan crude drug ysypó hû (A. marginatum) allowed the tentative identification of 20 constituents, including 9 iridoids, 6 phenolics, and 5 nitrogen-containing compounds. The main compounds were theviridoside and 4-hydroxy-1-methyl-L-proline. In addition, the presence of four chlortheviridosides is reported here for the first time. The present study showed that the crude drug and main compound were inactive towards PDE-5 in vitro. Nevertheless, the potential of ysypó hû as a male sexual enhancer cannot be discarded, since other mechanisms may be involved. Further in vitro and in vivo investigations are still needed to validate or refute the traditional use of ysypó hû as a male sexual enhancer.

Author Contributions

Participated in research design G.S.-H., R.F., F.J.-A., C.T.; Performed field work and collected data in the field R.F., C.C., N.S., S.V.; Conducted experiments F.J.-A, L.P., D.M.-C., C.T, A.B.-E.; Performed data analysis G.S.-H., A.B.-E., F.J.-A, D.M.-C, C.T. Wrote or contributed to the writing of the manuscript G.S.-H, A.B.-E., F.J.-A, C.T. All authors have read and agreed to the published version of the manuscript.

Funding

Consejo Nacional de Ciencia y Tecnología CONACYT, programa PRCOCIENCIA, recursos del Fondo para la Excelencia de la Educación e Investigación—FEEI del FONACIDE, project 14-INV-470 “Propiedades medicinales de plantas, conocimiento tradicional y patentes”, Paraguay, and PIEI-QUIM-BIO (Universidad de Talca).

Acknowledgments

We thank Leopoldo López and Belén Torres (CERI, Paraguay) for the patent search and advice on legal aspects of the project. Alberto Burgos-Edwards and Daniel Mieres-Castro thanks PFCHA-CONICYT for their doctoral grants number 2015-21151561 and 2017-21170479. Lisa Pormetter thanks DAAD-RISE program for financial support.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Müller, F. Drogas y medicamentos de los indios Guaraní (Mbyá, Pai y Chiripá) en las regiones orientales de la selva del Paraguay. Parodiana 1997, 10, 197–209. [Google Scholar]
  2. Kujawska, M. Yerba Mate (Ilex paraguariensis) beverage: Nutraceutical ingredient or conveyor for the intake of medicinal plants? Evidence from Paraguayan folk medicine. EBCAM 2018, 2018, 6849317. [Google Scholar]
  3. Soejarto, D.D. Ethnobotany of Stevia and Stevia rebaudiana. In The Genus Stevia; Kinghorn, D., Ed.; eBook; CRC Press: London, UK, 2001; pp. 1–224. [Google Scholar]
  4. Montenegro, P.; Quintana, R. Materia Medica Misionera: Herbolario Guaraní sigo XVII; Buena Vista Editores: Córdoba, Argentina, 2009. [Google Scholar]
  5. Arenas, P.; Moreno, A.R. Plants used as means of abortion, contraception, sterilization and fecundation by Paraguayan indigenous people. Econ. Bot. 1977, 31, 302–306. [Google Scholar] [CrossRef] [PubMed]
  6. Udulutsch, R.; Assis, M.A.; Dias, P. Taxonomic update of Adenocalymma (Bignoniaceae): Emendations, new synonyms, typification, and status change. Turk. J. Bot. 2013, 37, 630–643. [Google Scholar]
  7. Basualdo, I.; Soria, N.; Ortiz, M.; Degen, R. Uso medicinal de plantas comercializadas en los mercados de Asunción y Gran Asunción, Paraguay. Rev. Soc. Cient. Paraguay 2003, 14, 5–22. [Google Scholar]
  8. Basualdo, I.; Soria, N.; Ortiz, M.; Degen, R. Plantas comercializadas en los mercados de Asunción y Gran Asunción (Parte I). Rojasiana 2004, 6, 95–114. [Google Scholar]
  9. West, E.; Krychman, M. Natural aphrodisiacs—A review of selected sexual enhancers. Sex. Med. Rev. 2015, 3, 279–288. [Google Scholar] [CrossRef] [PubMed]
  10. Skalicka-Wozniak, K.; Georgiev, M.I.; Orhan, I.E. Adulteration of herbal sexual enhancers and slimmers: The wish for better sexual well-being and perfect body can be risky. Food Chem. Toxicol. 2017, 108, 355–364. [Google Scholar] [CrossRef] [PubMed]
  11. Gilard, V.; Balayssac, S.; Tinaugus, A.; Martins, N.; Martino, R.; Malet-Martino, M. Detection, identification and quantification by 1H NMR of adulterants in 150 herbal dietary supplements marketed for improving sexual performance. J. Pharm. Biomed. Anal. 2015, 102, 476–493. [Google Scholar] [CrossRef]
  12. Schmeda-Hirschmann, G.; Burgos-Edwards, A.; Theoduloz, C.; Jiménez-Aspee, F.; Vargas- Arana, G. Male sexual enhancers from the Peruvian Amazon. J. Ethnopharmacol. 2019, 229, 167–179. [Google Scholar] [CrossRef]
  13. Von Poser, G.L.; Schripsema, J.; Henriques, A.T.; Jensen, S.R. The distribution of iridoids in Bignoniaceae. Biochem. Syst. Ecol. 2000, 28, 351–366. [Google Scholar] [CrossRef]
  14. Ito, Y. Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. J. Chromatogr. A 2005, 1065, 145–168. [Google Scholar] [CrossRef] [PubMed]
  15. Abe, F.; Chen, R.-F.; Yamauchi, T.; Ohashi, H. Iridoids from the roots of Thevetia peruviana. Chem. Pharm. Bull. 1995, 43, 499–500. [Google Scholar] [CrossRef] [Green Version]
  16. Abe, F.; Yamauchi, T.; Yahara, S.; Nohara, R. Minor iridoids from Thevetia peruviana. Phytochemistry 1995, 38, 793–794. [Google Scholar] [CrossRef]
  17. Jones, G.P.; Naidu, B.P.; Waisel, Y.; Solomon, A.; Paleg, L.G. Occurrence and stress response of N-methylproline compounds in Tamarix species. Phytochemistry 2006, 67, 156–160. [Google Scholar] [CrossRef]
  18. Haraguchi, M.; Gorniak, S.L.; Ikeda, K.; Minami, Y.; Kato, A.; Watson, A.A.; Nash, R.J.; Molyneaux, R.J.; Asano, N. Alkaloidal componentes in the poisonous plant, Ipomoea carnea (Convolvulaceae). J. Agric. Food Chem. 2003, 51, 4995–5000. [Google Scholar] [CrossRef]
  19. Ren, L.; Xue, X.; Zhang, F.; Wang, Y.; Liu, Y.; Li, C.; Liang, X. Studies of iridoid glycosides using liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun. Mass Sp. 2007, 21, 3039–3050. [Google Scholar] [CrossRef]
  20. Noor, A.T.; Perveen, S.; Begum, A.; Fatima, I.; Malik, A.; Tareen, R.B. Aitchisonides A and B, new iridoid glucosides from Aitchisonia rosea. J. Asian Nat. Prod. Res. 2009, 11, 985–989. [Google Scholar] [CrossRef]
  21. Ono, M.; Ishimatsu, N.; Masuoka, C.; Yoshimitsu, H.; Tsuchihashi, R.; Okawa, M.; Kinjo, J.; Ikeda, T.; Nohara, T. Three new monoterpenoids from the fruit of Genipa americana. Chem. Pharm. Bull. 2007, 55, 632–634. [Google Scholar] [CrossRef] [Green Version]
  22. Es-Safi, N.-E.; Kerhoas, L.; Ducrot, P.H. Fragmentation study of iridoid glucosides through positive and negative electrospray ionization collision-induced dissociation and tandem mass spectrometry. Rapid Commun. Mass Sp. 2007, 21, 1165–1175. [Google Scholar] [CrossRef]
  23. Dinda, B.; Debnath, S.; Harigaya, Y. Naturally occurring iridoids. A review, part 1. Chem. Pharm. Bull. 2007, 55, 159–222. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Ling, S.-K.; Komorita, A.; Tanaka, T.; Fujioka, T.; Mihashi, K.; Kouno, I. Sulfur-containing bis iridoid glucosides and iridoid glucosides from Saprosma scortechinii. J. Nat. Prod. 2002, 65, 656–660. [Google Scholar] [CrossRef] [PubMed]
  25. Ling, S.-K.; Komorita, A.; Tanaka, T.; Fujioka, T.; Mihashi, K.; Kouno, I. Iridoids and anthraquinones from the Malaysian medicinal plant Saprosma scortchenii (Rubiaceae). Chem. Pharm. Bull. 2002, 50, 1035–1040. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Niu, X.; Xu, D.; Luo, J.; Kong, L. Main iridoid glycosides and HPLC/DAD-Q-TOF-MS/MS profile of glycosides from the antioxidant extract of Eucommia ulmoides Oliver seeds. Ind. Crops Prod. 2016, 79, 160–169. [Google Scholar] [CrossRef]
  27. Calis, I.; Kirmizibekmez, H.; Sticher, O. Iridoid glycosides from Globularia trichosantha. J. Nat. Prod. 2001, 64, 60–64. [Google Scholar] [CrossRef] [PubMed]
  28. Imakura, Y.; Kobayashi, S.; Yamahara, Y.; Kihara, M.; Tagawa, M.; Murai, F. Studies on constituents of Bignoniaceae plants. IV. Isolation and structure of a new iridoid glucoside, campsiside from Campsis chinensis. Chem. Pharm. Bull. 1985, 33, 2220–2227. [Google Scholar] [CrossRef] [Green Version]
  29. Martin, F.; Hay, A.-E.; Corno, L.; Gupta, M.P.; Hostettmann, K. Iridoid glycosides from the stems of Pithecoctenium crucigerum (Bignoniaceae). Phytochemistry 2007, 68, 1307–1311. [Google Scholar] [CrossRef]
  30. Buckingham, J. (Ed.) Dictionary of Natural Products on DVD; CRC Press, Taylor & Francis Group: Abingdon, UK, 2019. [Google Scholar]
  31. Chen, Y.-H.; Qi, J.; Hua, J.; Yu, B.-Y. Structural characterization and identification of major constituents in Radix Scrophulariae by HPLC coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Chin. J. Nat. Med. 2014, 12, 47–54. [Google Scholar]
  32. Li, L.; Tsao, R.; Liu, Z.; Liu, S.; Yang, R.; Young, J.C.; Zhu, H.; Deng, Z.; Xie, M.; Fu, Z. Isolation and purification of acteoside and isoacteoside from Plantago psyllium L. by high-speed counter-current chromatography. J. Chromatogr. A 2005, 1063, 161–169. [Google Scholar] [CrossRef]
  33. Pettit, G.R.; Numata, A.; Takemura, T.; Ode, R.H.; Narula, A.S.; Schmidt, J.M.; Cragg, G.M.; Pase, C.P. Antineoplastic agents, 107. Isolation of acteoside and isoacteoside from Castilleja linariaefolia. J. Nat. Prod. 1990, 53, 456–458. [Google Scholar] [CrossRef]
  34. Clifford, M.N.; Johnston, K.L.; Knight, S.; Kuhnert, N. Hierarchical scheme for LC-MSn identification of chlorogenic acids. J. Agric. Food Chem. 2003, 51, 2900–2911. [Google Scholar] [CrossRef] [PubMed]
  35. Winkler, T. Comments on piperidone derivatives from Dalbergia sympathetica. Magn. Reson. Chem. 2006, 44, 571–572. [Google Scholar] [CrossRef] [PubMed]
  36. De Aquino, P.E.; Magalhaes, T.R.; Nicolau, L.A.; Leal, L.K.; de Aquino, N.C.; Dos Santos, S.M.; Neves, K.R.; Silveira, E.R.; Viana, G.S. The anti-inflammatory effects of N-methyl-(2S,4R)-trans-4-hydroxy-L-proline from Sideroxylon obtusifolium are related to its inhibition of TNF-alpha and inflammatory enzymes. Phytomedicine 2017, 24, 14–23. [Google Scholar] [CrossRef] [PubMed]
  37. De Aquino, P.E.A.; de Souza, T.F.G.; Santos, F.A.; Viana, A.F.S.C.; Louchard, B.O.; Leal, L.K.A.M.; Rocha, T.M.; Evangelista, J.S.A.M.; de Aquino, N.C.; de Alencar, N.M.N.; et al. The wound healing property of N-methyl-(2S,4R)-trans-4-hydroxy-L-proline from Sideroxylon obtusifolium is related to its anti-inflammatory and antioxidant actions. J. Evid. Based Integr. Med. 2019, 24, 1–11. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  38. Zhang, P.K.; Chan, W.; Ang, I.L.; Wei, R.; Lam, M.M.T.; Lei, K.M.K.; Poon, T.C.W. Revisiting fragmentation reactions of protonated α-amino acids by high-resolution electrospray ionization tandem mass spectrometry with collision-induced dissociation. Sci. Rep. 2019, 9, 6453. [Google Scholar] [CrossRef] [PubMed]
  39. Torres, C.A.; Zampini, I.C.; Nuñez, M.B.; Isla, M.I.; Castro, M.P.; Gonzalez, A.M. In vitro antimicrobial activity of 20 selected climber species from the Bignoniaceae family. Nat. Prod. Res. 2013, 27, 2144–2148. [Google Scholar] [CrossRef]
  40. Granados-Echegoyen, C.; Pérez-Pacheco, R.; Alexander-Aguilera, A.; Lagunez-Rivera, L.; Alonso-Hernández, N.; Chairez-Martinez, E.d.J. Effects of aqueous and ethanol extract of dried leaves of Pseudocalymma alliaceum (Bignonaceae) on haematological and biochemical parameters of Wistar rats. Asian Pac. J. Reproduc. 2015, 4, 129–134. [Google Scholar] [CrossRef]
  41. Mendes, F.R. Tonic, fortifier and aphrodisiac: Adaptogens in the Brazilian folk medicine. Rev. Bras. Farmacog. 2011, 21, 754–763. [Google Scholar] [CrossRef] [Green Version]
  42. Corbin, J.D.; Francis, S.H. Molecular biology and pharmacology of PDE-5-inhibitor therapy for erectile dysfunction. J. Androl. 2003, 24, S38–S41. [Google Scholar] [CrossRef]
  43. Drewes, S.E.; George, J.; Khan, F. Recent findings on natural products with erectile-dysfunction activity. Phytochemistry 2003, 62, 1019–1025. [Google Scholar] [CrossRef]
  44. Kaufman, D.M. Neurologic aspects of sexual function. In Clinical Neurology for Psychiatrist; Kaufman, D.M., Ed.; Saunders: Philadelphia, PA, USA, 2007; pp. 355–370. [Google Scholar]
  45. Chen, Y.; Wu, Y.; Gan, X.; Liu, K.; Lv, X.; Shen, H.; Dai, G.; Xu, H. Iridoid glycoside from Cornus officinalis ameliorated diabetes mellitus-induced testicular damage in male rats: Involvement of suppression of the AGEs/RAGE/p38 MAPK signaling pathway. J. Ethnopharmacol. 2016, 194, 850–860. [Google Scholar] [CrossRef] [PubMed]
  46. Cicero, A.F.G.; Bandieri, E.; Arletti, R. Lepidium meyenii Walp. improves sexual behavior in male rats independently from its action on spontaneous locomotor activity. J. Ethnopharmacol. 2001, 75, 225–229. [Google Scholar] [CrossRef]
  47. Cicero, A.F.G.; Pacente, S.; Plaza, A.; Sala, E.; Arletti, R.; Pizza, C. Hexanic maca extract improves rat sexual performance more effectively than methanolic and chloroformic Maca extracts. Andrologia 2002, 34, 177–179. [Google Scholar] [CrossRef] [PubMed]
  48. Zenico, T.; Cicero, A.F.G.; Valmorri, L.; Mercuriali, M.; Bercovich, E. Subjective effects of Lepidium meyenii (Maca) extract on well-being and sexual performance in patients with mild erectile dysfunction: A randomized, double blind clinical trial. Andrologia 2009, 41, 95–99. [Google Scholar] [CrossRef] [PubMed]
Sample Availability: Samples of theviridoside are available from the authors.
Molecules 25 00180 g001 550
Figure 1. The Paraguayan crude drug “ysypó hû” (Adenocalymma marginatum). (A) flowering plant; (B) aerial parts; (C) root and stem.
Figure 1. The Paraguayan crude drug “ysypó hû” (Adenocalymma marginatum). (A) flowering plant; (B) aerial parts; (C) root and stem.
Molecules 25 00180 g001
Molecules 25 00180 g002 550
Figure 2. TLC of the counter-current chromatography (CCC) fractions of the Sephadex 8–15 of Adenocalymma marginatum (silica gel, EtOAc: formic acid: H2O: acetone 10:2:3:1).
Figure 2. TLC of the counter-current chromatography (CCC) fractions of the Sephadex 8–15 of Adenocalymma marginatum (silica gel, EtOAc: formic acid: H2O: acetone 10:2:3:1).
Molecules 25 00180 g002
Molecules 25 00180 g003 550
Figure 3. Structure of some of the main constituents of the Paraguayan crude drug ysypó hû (Adenocalymma marginatum). Arabic and roman numbers in parenthesis refer to Table 4.
Figure 3. Structure of some of the main constituents of the Paraguayan crude drug ysypó hû (Adenocalymma marginatum). Arabic and roman numbers in parenthesis refer to Table 4.
Molecules 25 00180 g003
Molecules 25 00180 g004 550
Figure 4. Ion chromatogram in the negative ionization mode (blue trace) and positive ionization mode (red trace), and HPLC profile at 280 nm of the root (a,c) and stem (b,d) extracts from the Paraguayan crude drug ysypó hû. Numbers refer to Table 4.
Figure 4. Ion chromatogram in the negative ionization mode (blue trace) and positive ionization mode (red trace), and HPLC profile at 280 nm of the root (a,c) and stem (b,d) extracts from the Paraguayan crude drug ysypó hû. Numbers refer to Table 4.
Molecules 25 00180 g004
Table
Table 1. Summary of the partition ratios (KD) values of biphasic solvent system (TBME/1-BuOH/ACN/H2O) for the main compounds in the Sephadex 8–15 fractions from the hydroalcoholic stem extract of Adenocalymma marginatum.
Table 1. Summary of the partition ratios (KD) values of biphasic solvent system (TBME/1-BuOH/ACN/H2O) for the main compounds in the Sephadex 8–15 fractions from the hydroalcoholic stem extract of Adenocalymma marginatum.
Molecules 25 00180 i001
System NumberProportions of TBME:n-BuOH:ACN:H2O +0.1% TFAKD Main Compound
11:3:1:5 *0.21
21:4:1:50.33
31:5:1:50.41
42:2:2.5:50.40
* Selected solvent system; U: upper organic phase; L: lower aqueous phase.
Table
Table 2. 1H, 13 C nuclear magnetic resonance (NMR) and heteronuclear multiple bond correlation (HBMC) data of theviridoside (compound 8) and its pentaacetate (400 and 100 MHz, respectively; MeOH-d4 or CDCl3, δ-values, J in. Hz).
Table 2. 1H, 13 C nuclear magnetic resonance (NMR) and heteronuclear multiple bond correlation (HBMC) data of theviridoside (compound 8) and its pentaacetate (400 and 100 MHz, respectively; MeOH-d4 or CDCl3, δ-values, J in. Hz).
1H13C1H13CHMBC
MeOH-d4MeOH-d4CDCl3CDCl3CDCl3
15.55 d (5.6)95.67 d5.32 d (4.4)95.26 d
37.52 s152.71 d7.31 s151.36 d166.63, 113.78, 95.26, 74.91
4-113.14 s-113.78 s
5-75.12 s-74.91 s5.32
62.85 br s45.54 t2.75 d (19.6)
2.80 d (19.6)		45.54 t129.81, 134.68, 74.91
75.72 s125.44 d5.68 br s129.81 d
8-140.59 s-134.68 s
93.06 d (6.0)55.37 d3.08 d (2.4)54.94 d
104.25 d (14.4)
4.14 d (14.4)		59.63 t4.58 d (13.6)
4.51 d (13.6)		61.44 t
11-166.96 s-166.63 s
OMe3.75 s50.41 q3.67 s51.41 q
1’4.65 d (8.0)98.66 d4.73 d (8)96.51 d
2’3.25 dd (8.8, 8.0)73.18 d4.91 t (8.8)70.73 d
3’3.33 m77.03 d5.16 t (9.6)72.03 d
4’3.42 m76.18 d5.00 t (9.6)68.15 d
5’3.32 m 70.15 d3.67 m72.10 d
6’3.83-3.85 m,
3.60-3.65 m		62.87 t4.17 dd (12.4, 4)
4.07 dd (12.4, 1)		61.44 t
Ac
COO---170.51 s (2C), 170.05 s, 169.76 s, 169.27 s
CH3--2.00 s, 1.98 s, 1.93 s, 1.91 s, 1.89 s20.74 q, 20.62 q, 20.51 q (2C), 20.39 q
Table
Table 3. 1H- and 13C-NMR data of 4-Hydroxy-1-methyl-l-proline (compound I)(400 and 100 MHz, respectively; MeOH-d4, δ-values, J in Hz).
Table 3. 1H- and 13C-NMR data of 4-Hydroxy-1-methyl-l-proline (compound I)(400 and 100 MHz, respectively; MeOH-d4, δ-values, J in Hz).
HCHMBCConfiguration
1-170.38 s4.36, 3.08, 2.27
24.36 dd (11.2, 7.6)69.03 d3.88, 3.16, 3.08, 2.27S
32.50 ddt (14, 7.2, 1.6),
2.27 ddd (14, 11.2, 4.8)		38.48 t
44.55 m68.84 d R
53.88 dd (12.4, 4.4),
3.16 dt (12.4, 2)		63.29 t3.08
N-CH33.08 s42.79 q3.88, 4.36; 63.29, 68.84
Table
Table 4. Tentative identification of compounds from root and stem hydroalcoholic extracts of Adenocalymma marginatum by HPLC-ESI-MS/MS in the negative ion mode and distribution in roots and stems.
Table 4. Tentative identification of compounds from root and stem hydroalcoholic extracts of Adenocalymma marginatum by HPLC-ESI-MS/MS in the negative ion mode and distribution in roots and stems.
PeakRt (min)[M + H]+/[M − H]− m/zMS/MS (%)Tentative IdentificationRootsStems
[M + H]+
I6.0145.999.50 (100)4-Hydroxy-1-methyl-l-proline *XX
II6.099.681.5(100)1-methyl-2,3-dihydro-3-hydroxypyrrol-X
III6.181.5 1-methylpyrrolXX
IV6.4115.770.0(100), 28.1 (23), 43.1(20)l-prolineXX
V15.3131.986.1 (100), 69.1(45), 56.9(4)l-isoleucine *XX
[M − H]−
15.5340.9 178.4(100)Caffeoyl hexoside XX
29.0727.2564.8(100), 341(14), 240.5(3)Theviridoside dihexoside 1-X
311.2763.3727.0 (100), 484.9 (29), 383.1 (43), 340.7 (29), 240.6 (11)Chlortheviridoside dihexoside derivative-X
412.3601.4564.9 (100), 385.1 (73), 301 (27), 240.6 (4), 222.6 (15)Chlortheviridoside hexoside derivativeXX
512.6726.9502.5(42), 385.1 (73), 240.5(3)Theviridoside dihexoside 2XX
613.5601.2564.9 (100), 385.1 (13), 240.7 (5)Chlortheviridoside hexoside derivativeXX
713.6659.1402.3(93), 254.9(78), 240.6(100)Methyl-theviridoside aglycon theviridoside X-
815.7403.4240.67(100), 222.60(21)Theviridoside* XX
915.9-16.2600.9565.0 (100), 240.8 (13)Chlortheviridoside hexoside derivativeXX
1016.1-16.3565.5240.65(100)Theviridoside hexosideXX
1139.4625.6461.2(100), 315.3(3)Dihydro acteoside-X
1241.3769.6607.3(100), 461.2(2)Angoroside B XX
1341.7–42.1623.6477.1(4), 461.2(100), 315.2(2)ActeosideXX
1443.8–45.5623.5461.1(100), 315.2(2), 178.6(3) IsoacteosideXX
1551.3515.7352.9(100), 172.6(7)Dicaffeoylquinic acid-X
* Identity confirmed by NMR spectroscopy analysis—not detected.

Share and Cite

MDPI and ACS Style

Schmeda-Hirschmann, G.; Burgos-Edwards, A.; Jiménez-Aspee, F.; Mieres-Castro, D.; Theoduloz, C.; Pormetter, L.; Fogel, R.; Céspedes, C.; Soria, N.; Valdez, S. Iridoids and Amino Acid Derivatives from the Paraguayan Crude Drug Adenocalymma marginatum (ysypó hû). Molecules 2020, 25, 180. https://doi.org/10.3390/molecules25010180

AMA Style

Schmeda-Hirschmann G, Burgos-Edwards A, Jiménez-Aspee F, Mieres-Castro D, Theoduloz C, Pormetter L, Fogel R, Céspedes C, Soria N, Valdez S. Iridoids and Amino Acid Derivatives from the Paraguayan Crude Drug Adenocalymma marginatum (ysypó hû). Molecules. 2020; 25(1):180. https://doi.org/10.3390/molecules25010180

Chicago/Turabian Style

Schmeda-Hirschmann, Guillermo, Alberto Burgos-Edwards, Felipe Jiménez-Aspee, Daniel Mieres-Castro, Cristina Theoduloz, Lisa Pormetter, Ramon Fogel, Claudia Céspedes, Nelida Soria, and Sintya Valdez. 2020. "Iridoids and Amino Acid Derivatives from the Paraguayan Crude Drug Adenocalymma marginatum (ysypó hû)" Molecules 25, no. 1: 180. https://doi.org/10.3390/molecules25010180

APA Style

Schmeda-Hirschmann, G., Burgos-Edwards, A., Jiménez-Aspee, F., Mieres-Castro, D., Theoduloz, C., Pormetter, L., Fogel, R., Céspedes, C., Soria, N., & Valdez, S. (2020). Iridoids and Amino Acid Derivatives from the Paraguayan Crude Drug Adenocalymma marginatum (ysypó hû). Molecules, 25(1), 180. https://doi.org/10.3390/molecules25010180

Article Metrics

Back to TopTop