Communication Temporal Variation of chilensis Aristolochic Acids during Spring

Rocío Santander *, Alejandro Urzúa *, Ángel Olguín and María Sánchez

Received: 7 October 2015 ; Accepted: 9 November 2015 ; Published: 13 November 2015 Academic Editor: Derek J. McPhee

Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40, Correo 33, Santiago 9170022, Chile; [email protected] (Á.O.); [email protected] (M.S.) * Correspondence: [email protected] (R.S.); [email protected] (A.U.); Tel.: +56-2-2718-1155 (R.S.); +56-2-2718-1154 (A.U.)

Abstract: In this communication, we report the springtime variation of the composition of aristolochic acids (AAs) in Aristolochia chilensis leaves and stems. The dominant AA in the leaves of all samples, which were collected between October and December, was AA-I (1), and its concentration varied between 212.6 ˘ 3.8 and 145.6 ˘ 1.2 mg/kg and decreased linearly. This decrease occurred in parallel with the increase in AA-Ia (5) concentration from 15.9 ˘ 0.8 mg/kg at the beginning of October to 96.8 ˘ 7.8 mg/kg in mid-December. Both acids are enzymatically related by methylation-demethylation reactions. Other AAs also showed important variations: AA-II (2) significantly increased in concentration, reaching a maximum in the first two weeks of November and subsequently decreasing in mid-December to approximately the October levels. The principal component in the AA mixture of the stems was also AA-I (1); similar to AA-II (2), its concentration increased beginning in October, peaked in the second week of November and subsequently decreased. The concentrations of AA-IIIa (6) and AA-IVa (7) in the leaves and stems varied throughout the study period, but no clear pattern was identified. Based on the variation of AAs in A. chilensis leaves and stems during the study period, the reduced contents of non-phenolic AAs and increased concentrations of phenolic AAs are likely associated with a decrease in this ’s toxicity during the spring.

Keywords: Aristolochia chilensis; aristolochic acids; HPLC-DAD; temporal variation

1. Introduction Species of the genus Aristolochia () have been used in folk medicine worldwide to treat various diseases [1]. Aristolochia contains aristolochic acids (AAs), a group of aporphinoids (10-nitrophenanthrene-1-carboxylic acids). Among the known AAs, AA-I (1) and AA-II (2) Figure1, are powerful carcinogens in mice, rats and humans. Studies have shown that these AAs are genotoxic, mutagenic, and nephrotoxic [1,2]. Two species represent the family Aristolochiaceae in Chile: Aristolochia chilensis Bridges ex Lindl. and Aristolochia bridgesii (Klotzsch) Duch. The former is a summer-deciduous, low-creeping herb that ranges southwards from Caldera in Northern Chile (27˝S) to beyond the latitude of Santiago (34˝S), and it is known by the local name of “hierba de la Virgen María” (Virgin Mary’s herb) [3]. Teas from the leaves and, particularly, the roots of Aristolochia chilensis Bridges ex Lindl. have been used in Central Chile since the 19th century as an anti-hemorrhagic agent and to expel the residual placenta after childbirth [4,5]. In Chilean folk medicine, the common name “Virgin Mary’s herb” of A. chilensis is also given to two other medicinal (Stachys albicaulis Lindl. and Phyla nodiflora (L.) Greene). This coincidence

Molecules 2015, 20, 20391–20396; doi:10.3390/molecules201119704 www.mdpi.com/journal/molecules Molecules 2015, 20, 20391–20396 of vernacular names has resulted in a consumption of the aerial parts of A. chilensis that is much higher than expectedMolecules because 2015, 20, page–page of the confusion of herbalists in rural markets, who may have no botanical training [6,7]. Although the risk of using A. chilensis in folk medicine is evident, the plant continues botanical training [6,7]. Although the risk of using A. chilensis in folk medicine is evident, the plant to be used.continues to be used.

Figure 1. Aristolochic acids in Aristolochia chilensis. Figure 1. Aristolochic acids in Aristolochia chilensis. A recent study has shown that the roots of Aristolochia chilensis contain a mixture of AA-I (1), AA-II (2), AA-III (3), AA-IV (4), AA-Ia (5), AA-IIIa (6), AA-IVa (7), and aristoloside (8) (a A recent study has shown that the roots of Aristolochia chilensis contain a mixture of AA-I 6-O-β-D-glucopyranoside of AA-IVa (7)). This mixture is rich in phenolic AAs and AA-IVa (7), and (1), AA-IIaristoloside (2), AA-III (8) accounts (3), AA-IV for approximately (4), AA-Ia 90% ( 5of), the AA-IIIa total AAs. ( 6In), contrast, AA-IVa the most (7), toxic and compound, aristoloside (8) (a 6-O-β-D-glucopyranosideAA-I (1), is a minor component of AA-IVa [8]. (Previous7)). This investigations mixture of is the rich aerial in parts phenolic of A. chilensis AAs revealed and AA-IVa (7), and aristolosidethe presence (8) accountsof a mixture for of approximatelyAA-I (1), AA-II (2), 90% AA-Ia of (5 the), AA-IIIa total ( AAs.6) and AA-IVa In contrast, (7) and thetrace most toxic amounts of AA-III (3) and AA-IV (4) [9]. In contrast to the roots, the main AA component of the compound, AA-I (1), is a minor component [8]. Previous investigations of the aerial parts of A. aerial parts of A. chilensis is AA-I (1), which accounts for approximately 50% of the total AAs [8]. chilensis revealedBecause the most presence toxic AA of is a mixtureAA-I (1) [10], of AA-I human (1 consumption), AA-II (2), of AA-Ia the aerial (5), parts AA-IIIa of A. chilensis (6) and is AA-IVa (7) and trace amountslikely more of dangerous AA-III (than3) and the consumption AA-IV (4)[ of9]. the In root. contrast to the roots, the main AA component of the aerial partsAristolochia of A. chilensis chilensis isblooms AA-I from (1), mid-August which accounts to early September, for approximately reaches the peak 50% of of vegetative the total AAs [8]. development in late November, and declines during December, at which time, the aerial parts are Because the most toxic AA is AA-I (1)[10], human consumption of the aerial parts of A. chilensis is lost. Because of the increasing concern caused by the indiscriminate consumption of A. chilensis, we likely moreevaluated dangerous the variation than the in the consumption AA content and of compos the root.ition in the leaves and stems of a population of Aristolochiathis plant chilensis during harvestingblooms from by herbalists. mid-August In this tostudy, early high-performance September, reaches liquid chromatography the peak of vegetative developmentwith indiode-array late November, detection (HPLC-DAD), and declines which during is a widely December, used technique at which to detect time, and the quantify aerial parts are AAs in herbal medicine [8,9,11–16], was used to determine the amounts of AA-I (1), AA-II (2), AA-Ia lost. Because of the increasing concern caused by the indiscriminate consumption of A. chilensis, we (5), AA-IIIa (6) and AA-IVa (7) from samples of A. chilensis leaves and stems, which were collected evaluatedduring the variation spring. in the AA content and composition in the leaves and stems of a population of this plant during harvesting by herbalists. In this study, high-performance liquid chromatography with diode-array2. Resultsdetection and Discussion (HPLC-DAD), which is a widely used technique to detect and quantify AAs in herbalWe medicine determined [8, 9the,11 contents–16], was of AA-I used (1), to AA-II determine (2), AA-Ia the (5), amounts AA-IIIa (6 of) and AA-I AA-IVa (1), ( AA-II7) from (2), AA-Ia (5), AA-IIIasamples (6) and of A. AA-IVa chilensis ( 7leaves) from and samples stems, which of A. were chilensis collectedleaves in spring and stems,between which October were and collected December. The results are summarized in Tables 1 and 2. during spring.The dominant AA in the leaves of all collected samples between October and December was AA-I (1); AA-I(1) clearly decreases of 25% but still his content is 30% of the total AAs in December, 2. ResultsPearson and Discussion coefficient (r2 −0.9513), whereas AA-Ia (5) increases linearly of 20% and showed a positive Pearson coefficient (r2 0.8679), Figure 2. We determinedThis increase the occurred contents in parallel of AA-I with ( the1), decrease AA-II in (2 the), AA-IaAA-I (1) (concentration.5), AA-IIIa Because (6) and AA-I AA-IVa (7) from samples(1) and of AA-IaA. chilensis (5) are relatedleaves by andsimple stems, enzymatic which methylat wereion-demethylation collected in spring reactions, between the decrease October and December.in The the resultsAA-I (1)are concentration summarized is directly in Tables relate1d and to the2. increase in the AA-Ia (5) concentration. The dominantIn addition, AAAA-II in (2) the can leavesbe hydroxylated of all collected to AA-Ia (5 samples) and AA-IIIa between (6), also Octobershows a weak and positive December was Pearson coefficient (r2 0.6986). AA-I (1) has been found to be severely cytotoxic and elicits maximal AA-I (1); AA-I(1) clearly decreases of 25% but still his content is 30% of the total AAs in December, toxicity in renal epithelial cells (IC50 value: 10 mmol/L), whereas AA-Ia (5) is twenty times less toxic Pearson coefficient(IC50 value: 200 (r2 mmol/L)´0.9513), [10]. whereas AA-Ia (5) increases linearly of 20% and showed a positive Pearson coefficient (r2 0.8679), Figure2. 2 This increase occurred in parallel with the decrease in the AA-I (1) concentration. Because AA-I (1) and AA-Ia (5) are related by simple enzymatic methylation-demethylation reactions, the decrease in the AA-I (1) concentration is directly related to the increase in the AA-Ia (5) concentration. In addition, AA-II (2) can be hydroxylated to AA-Ia (5) and AA-IIIa (6), also shows a weak positive Pearson coefficient (r2 0.6986). AA-I (1) has been found to be severely cytotoxic and elicits maximal

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toxicity in renal epithelial cells (IC50 value: 10 mmol/L), whereas AA-Ia (5) is twenty times less toxic (IC50 value: 200 mmol/L) [10]. No correlation was found in the variation of AA-II (2) and AA-IVa (7) (Figure2). The AA contentsMolecules in the 2015 stems, 20, page–page of the collected samples between October and December showed no normality (Shapiro-Wilk test). However, from October to December, the concentrations of AA-IIIa (6) and No correlation was found in the variation of AA-II (2) and AA-IVa (7) (Figure 2). The AA AA-IVacontents (7) increased in the stems by 7.4of the and collected 16.8 times,samples respectively. between October Additionally, and December AA-Ia showed ( 5no) wasnormality detected in the November(Shapiro-Wilk and December test). However, samples from (Table October2). to December, the concentrations of AA-IIIa (6) and AA-IVa (7) increased by 7.4 and 16.8 times, respectively. Additionally, AA-Ia (5) was detected in the TableNovember 1. Content and (mg/kg,December X samples˘ SD) of (Table AA-I 2). (1 ), AA-II (2), AA-Ia (3), AA-IIIa (4) and AA-IVa (5) from samples of leaves of A. chilensis collected between October and December 2014. Table 1. Content (mg/kg, X ± SD) of AA-I (1), AA-II (2), AA-Ia (3), AA-IIIa (4) and AA-IVa (5) from samples of leaves of A. chilensis collected between October and December 2014. Non Phenolic Acids Phenolic Acids Harvest Date AA-INon Phenolic AA-II Acids AA-IaPhenolic AA-IIIa Acids AA-IVa Total AAs October-15Harvest Date 212.6 ˘ 3.8 AA-I 71.6 ˘AA-II2.2 15.9AA-Ia˘ 0.8 AA-IIIa 41.2 ˘ 3.7AA-IVa 46.7 Total˘ 0.8 AAs 388.0 ˘ 5.9 November-1October-15 206.3 ˘ 212.65.8 ± 3.8 146.8 71.6˘ 5.9 ± 2.2 15.9 7.7 ˘± 0.81.0 41.2 56.3± 3.7˘ 3.2 46.7 ± 0.8 36.9 388.0˘ 2.4 ± 5.9 453.9 ˘ 9.2 November-15November-1 195.5 ˘ 206.32.8 ± 5.8 144.6 146.8˘ 2.4 ± 5.9 26.97.7 ±˘ 1.00.3 56.3 104.9 ± 3.2˘ 3.2 36.9 ± 2.4 61.5 453.9˘ 2.8 ± 9.2 533.5 ˘ 5.7 November-30November-15 154.5 ˘ 195.50.8 ± 2.8 129.7 144.6˘ 3.1 ± 2.4 26.9 61.9 ±˘ 0.38.1 104.9 49.4 ± 3.2˘ 9.5 61.5 ± 2.8 31.4 533.5˘ 4.7 ± 5.7 426.9 ˘ 13.7 ˘ ˘ ˘ ˘ ˘ ˘ December-15November-30 145.6 154.51.2 ± 0.8 78.3 129.70.5 ± 3.1 61.9 96.8 ± 8.17.8 49.4 96.8± 9.5 7.8 31.4 ± 4.7 61.6 426.97.1 ± 13.7 479.1 13.2 December-15 145.6 ± 1.2 78.3 ± 0.5 96.8 ± 7.8 96.8 ± 7.8 61.6 ± 7.1 479.1 ± 13.2 Table 2. Content (mg/kg, X ˘ SD) of AA-I (1), AA-II (2), AA-Ia (3), AA-IIIa (4) and AA-IVa (5) from samplesTable of stems 2. Content of A. (mg/kg, chilensis Xcollected ± SD) of AA-I between (1), AA-II October (2), AA-Ia and ( December3), AA-IIIa (4 2014.) and AA-IVa (5) from samples of stems of A. chilensis collected between October and December 2014.

NonNon Phenolic Phenolic Acids Acids Phenolic Phenolic Acids Acids Harvest DateHarvest Date AA-I AA-I AA-IIAA-II AA-Ia AA-Ia AA-IIIa AA-IIIa AA-IVa AA-IVaTotal-AAs Total-AAs October-15October-15 104.6 ˘ 104.63.2 ± 3.2 20.6 20.6˘ 0.7 ± 0.7 - - 12.3 ± 12.3 2.5˘ 2.5 5.4 ± 1.0 5.4 142.8˘ 1.0 ± 4.2 142.8 ˘ 4.2 November-1November-1 307.5 ˘ 307.53.0 ± 3.0 68.4 68.4˘ 1.5 ± 1.5 - - 39.2 ± 39.2 2.0˘ 2.0 23.7 ± 2.2 23.7 438.8˘ 2.2 ± 4.5 438.8 ˘ 4.5 November-15November-15 325.3 ˘ 325.38.2 ± 8.2 118.6 118.6˘3.7 ±3.7 - - 49.3 ± 49.3 2.0˘ 2.0 47.1 ± 2.9 47.1 540.3˘ 2.9 ± 9.7 540.3 ˘ 9.7 November-30November-30 294.6 ˘10.6 294.6±10.6 102.1 102.1˘2.9 ±2.9 8.8 8.8 ±˘ 0.50.5 41.2 ± 41.2 3.0˘ 3.0 39.4 ± 3.0 39.4 486.1˘ 3.0 ± 11.8 486.1 ˘ 11.8 December-15 265.4 ˘ 3.7 65.5 ˘ 3.4 46.9 ˘ 3.6 90.6 ˘ 9.2 90.6 ˘ 9.8 559.0 ˘ 14.8 December-15 265.4 ± 3.7 65.5 ± 3.4 46.9 ± 3.6 90.6 ± 9.2 90.6 ± 9.8 559.0 ± 14.8

Figure 2. Variation during springtime of the percentage (%) of each aristolochic acid (AA), in the Figure 2. Variation during springtime of the percentage (%) of each aristolochic acid (AA), in the total total AAs mixture from leaves samples. AAs mixture from leaves samples. 3. Experimental Section 3. Experimental Section 3.1. Plant Material 3.1. Plant MaterialFieldwork was performed at Cuesta Lo Prado (15 km west of Santiago, 33°28′S, 70°56′W, 750 m above sea level) from mid-October to mid-December. Thirty Aristolochia chilensis plants of similar Fieldwork was performed at Cuesta Lo Prado (15 km west of Santiago, 33˝281S, 70˝561W, 750 m 3 above sea level) from mid-October to mid-December. Thirty Aristolochia chilensis plants of similar

20393 Molecules 2015, 20, 20391–20396 sizes and phenological stages were chosen and marked (N˝ 1–30). From this group, 5 plants were randomlyMolecules selected 2015, 20, page–page every fifteen days. Leaf and stem samples of each of the five plants were collected and immediately oven-dried at 50 ˝C for 24 h, milled and subjected to extraction and analysis. sizes and phenological stages were chosen and marked (N° 1–30). From this group, 5 plants were 3.2. Extractrandomly Preparation selected every fifteen days. Leaf and stem samples of each of the five plants were collected and immediately oven-dried at 50 °C for 24 h, milled and subjected to extraction and analysis. Each oven-dried and powdered sample (20 g) was extracted with methanol (160 mL) at room temperature3.2. Extract for Preparation 24 h and subsequently boiled for 4 h. The suspension was vacuum filtered, and the plant material was washed with methanol (40 mL). The combined extract was evaporated in vacuo. Each oven-dried and powdered sample (20 g) was extracted with methanol (160 mL) at room The syrupy residue was agitated with 60 mL of 5% NaHCO at 40 ˝C, allowed to stand at room temperature for 24 h and subsequently boiled for 4 h. The suspension3 was vacuum filtered, and the temperatureplant material and washed was washed with with CHCl methanol3 (4 ˆ 15 (40 mL) mL). and The ethyl combined acetate extract (4 ˆ was15 mL). evaporated Washing in vacuo with. CHCl3 and AcOEtThe after syrupy evaporation residue was yielded agitated brownwith 60 ormL dark-green of 5% NaHCO extracts3 at 40 that°C, allowed contained to stand no acids,at room which weretemperature not investigated and washed further. with The CHCl aqueous3 (4 × 15 phase mL) and was ethyl adjusted acetate to (4 pH × 15 2 usingmL). Washing HCl and with extracted CHCl3 with ethyland acetate AcOEt (4 afterˆ 15 evaporation mL). Evaporation yielded brown of the or combined dark-green extracts extractsin that vacuo containedyielded no a fractionacids, which of crude AAs.were The not procedure investigated was further. repeated The aqueous for 25 samplesphase wa (20s adjusted g) of leavesto pH 2 andusing 25 HCl (20 and g) extracted samples with of stems, whichethyl were acetate obtained (4 × 15 from mL). five Evapor differentation of plants the combined on each collectionextracts in date.vacuo yielded a fraction of crude AAs. The procedure was repeated for 25 samples (20 g) of leaves and 25 (20 g) samples of stems, 3.3. HPLCwhich Analysiswere obtained of AAs from five different plants on each collection date.

3.3.The HPLC crude Analysis AA fractions of AAs were analyzed using HPLC (Waters 600; Milford, MA, USA) with a reverse-phase Symmetry Shield RP18 column (5-µm particle size; 25 ˆ 0.46 cm). The method was The crude AA fractions were analyzed using HPLC (Waters 600; Milford, MA, USA) with a describedreverse-phase in a previous Symmetry paper Shield [16]. RP18 Briefly, column gradient (5-μm elution particle was size; performed 25 × 0.46 cm). as follows The method using was a mobile phase,described which in consisted a previous of paper 0.1% [16]. acetic Briefly, acid gradient in water elution (solution was A)performed and 0.1% as follows acetic acidusing in a mobile acetonitrile (solutionphase, B): which 0–5 min, consisted isocratic of 0.1% elution acetic with acid 70% in water A/30% (solution B; 5–45 A) min,and 0.1% linear acetic gradient acid in from acetonitrile 70% A/30% B to 55%(solution A/45% B): 0–5 B. min, A Waters isocratic 2996 elution diode-array-detector with 70% A/30% B; (DAD)5–45 min, was linear used gradient to detect from the 70% AAs, A/30% and B their spectrato 55% were A/45% recorded B. A Waters at wavelengths 2996 diode-array-detec of 200–800tor nm. (DAD) The was UV used spectra to detect and the retention AAs, and times their of all detectedspectra AAs were were recorded coincident at wavelengths with the AA-Iof 200–800 (1), AA-IInm. The (2 ),UV AA-Ia spectra (5), and AA-IIIa retention (6) times and AA-IVaof all (7) standards,detected Figure AAs 3were[8,9]. coincident Quantification with the was AA-I based (1), AA-II on the (2 areas), AA-Ia of the(5), peaksAA-IIIa in (6 the) and chromatograms, AA-IVa (7) standards, Figure 3 [8,9]. Quantification was based on the areas of the peaks in the chromatograms, which were determined at 254 nm. A dilution series of standard solutions was prepared from stock which were determined at 254 nm. A dilution series of standard solutions was prepared from stock solutions of the standards and all standard and sample solutions were stored at 5 ˝C. Calibration solutions of the standards and all standard and sample solutions were stored at 5 °C. Calibration curvescurves were were obtained obtained by by plotting plotting the the peakpeak areasareas against against the the standard standard concentrations; concentrations; these these curves curves werewere used used to determine to determine the the AA AA concentrations concentrations inin the samples. samples.

Figure 3. Representative chromatograms of leaves and stems aristolochic acids from Aristolochia chilensis. Figure 3. Representative chromatograms of leaves and stems aristolochic acids from Aristolochia chilensis.

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3.4. Statistical Analysis The Shapiro-Wilk test was used in data normality testing. Linear correlations between the concentrations of AAs and the dates when the plant samples were collected were assessed by calculating the Pearson correlation coefficient.

4. Conclusions The findings reveal that the AA composition of Aristolochia chilensis leaves changes throughout spring, exhibiting a linear decrease in the concentration of the non-phenolic acid AA-I (1) and linear increases in the concentration of the phenolic acids AA-Ia (5) and AA-IIIa (6). The variations of AA-II (2) and AA-IVa (7) were not adjusted to a linear correlation model. However, from October to December, the total concentration of phenolic AAs increased, and the concentration of non-phenolic AAs decreased. The AA concentrations in the stems collected between October and December showed no normality (Shapiro-Wilk test). Based on the variation of the AAs in A. chilensis leaves and stems during the study period, the reduced content of non-phenolic AAs and the increased concentration of phenolic AAs are likely associated with a decrease of this plant’s toxicity during the spring.

Acknowledgments: This work was supported by Grant No. 1120037 (FONDECYT) and the Universidad de Santiago de Chile, Proyectos Basales No. 02154UM and Vicerrectoría de Investigación, Desarrollo e Innovación Usach. Author Contributions: A.U. designed the study and wrote the paper, R.S. performed the experiments, and M.S. and A.O. performed the data analysis. Conflicts of Interest: The authors declare no conflict of interest.

References

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