Phytotoxic Compounds from Xanthocephalum gymnospermoides var. eradiatum ¹

• Abstract
• Introduction
• Materials and Methods
• General experimental procedures
• Plant material
• Phytogrowth-inhibitory bioassays
• Extraction and preliminary fractionation
• Isolation of 8α,13S-epoxylabdane-14S,15-diol (1), methyl grindelate (2), grindelic acid (3) and 4,5-epoxy-β-caryophyllene (7)
• Isolation of 7α,8α-epoxygrindelic acid (4), 7α-hydroxy-8(17)-dehydrogrindelic acid (5) and 17-hydroxygrindelic acid (6) as their methyl esters 4 a, 5 a and 6 a, respectively
• Physical and spectral properties of 8α,13S-epoxylabdane-14S,15-diol (1)
• Physical and spectral properties of methyl grindelate (2)
• Results and Discussion
• Acknowledgements
• References

Abstract

Investigation of the aerial parts of Xanthocephalum gymnospermoides var eradiatum led to the isolation of two new labdane-type of diterpenes, namely, 8α,13S-epoxylabdane-14S,15-diol (1) and methyl grindelate (2). In addition, grindelic acid (3), 7α,8α-epoxygrindelic acid (4), 7α-hydroxy-8(17)dehydrogrindelic acid (5), 17-hydroxygrindelic acid (6) and 4,5-epoxy-β-caryophyllene (7) were obtained. The isolated compounds were characterized by spectral means. The absolute configuration of compound 1 was established by chemical correlation with 8α,13S-epoxy-15-nor-labdan-14-oic acid methyl ester of known absolute stereochemistry and by using the advanced Mosher's ester methodology. The results of the present investigation indicated that the known compound barbatol (8) could be an enantiomer of compound 1. Compounds 1 - 3 and 7 caused significant inhibition of the radicle growth of seedlings of Amaranthus hypochondriacus.

Introduction

Xanthocephalum gymnospermoides (A. Gray) B. & H. var. eradiatum Lane (Asteraceae) is an endemic yellow flowered, annual herb restricted to the mountain valleys of western Chihuahua, México. In this habitat the variety forms dense populations along the margins of cultivated and fallowed fields being the dominant plant. X. gymnospermoides var eradiatum often excludes other annual weeds such as species of the genera Amaranthus, Bidens and Chenopodium. This field observation suggests the presence of allelopathic compounds in the plant. The Tarahumara Indians call the plant “uriso” and attribute no contemporary use to it although in the past the crushed fresh leaves were applied to heal skin sores. To the best of our knowledge, this plant has not been the subject of phytochemical analysis, but the genus Xanthocephalum has yielded some flavonoids [1], diterpenoids, triterpenoids, phenylpropanoids and acetylenes [2].

In the course of our continuing search for plant-growth inhibiting agents from Mexican medicinal plants we describe in this investigation the isolation and characterization of the phytotoxic principles from the aerial parts of X. gymnospermoides var. eradiatum.

Materials and Methods

General experimental procedures

Melting point determinations were performed on a Fisher-Johns apparatus and are uncorrected. Optical rotations were taken on a digital polarimeter JASCO Dip 360. IR spectra (KBr) were obtained on a Perkin-Elmer 599 B spectrophotometer. NMR spectra including COSY, NOESY, HMQC and HMBC experiments, were recorded on a Varian VXR-300 S or a Varian UNITY PLUS 500 spectrometers in CDCl₃ either at 500 (¹H) or 125 (¹³C) MHz, using tetramethylsilane (TMS) as internal standard. Mass spectra were registered in a JEOL JMS-AX505 HA mass spectrometer. EI mass spectra were obtained using ionization voltage of 70 eV. Analytical and preparative TLC were performed on pre-coated silica gel 60 F254 plates (Merck). TLC spots were visualized by spraying with a 10 % solution of Ce(SO₄)₂ in 2 N H₂SO₄, followed by heating at 110 °C. For open CC, silica gel 60 (70 - 230 mesh, Merck) was used.

Plant material

The aerial parts of X. gymnospermoides var. eradiatum were collected in Municipio de Guachochic, State of Chihuahua, México, in September 1994. A voucher specimen of the plant (Bye 19286) is preserved in the Ethnobotanical collection of the National Herbarium (MEXU), Instituto de Biología, UNAM.

Phytogrowth-inhibitory bioassays

The phytogrowth-inhibitory activity of the extract and pure compounds was evaluated on seeds of Amaranthus hypochondriacus by using the Petri dish bioassay (PDB) [3]. In addition, a direct bioautographic bioassay system (BPIB) was employed to guide secondary fractionation and speed up the isolation of active compounds [3]. The data were analyzed by ANOVA (P < 0.05). The extract and pure compounds were tested at 10, 100 and 1000 μg ml^-1. 2,4-D was used as the positive control. The bioassays were performed at 28 °C.

Extraction and preliminary fractionation

The air-dried plant material (2 kg) was ground into powder and extracted exhaustively by maceration at room temperature with CHCl₃/MeOH, 1 : 1. After filtration, the extract was concentrated in vacuo to yield 142 g of residue. The active extract was subjected to column chromatography (CC) over silica gel (900 g) and eluted with a gradient of hexane/CHCl₃/MeOH. One hundred seventy eight fractions (1 l each) were collected and pooled based on their TLC profiles to yield nine mayor fractions (FI-FIX).

Isolation of 8α,13S-epoxylabdane-14S,15-diol (1), methyl grindelate (2), grindelic acid (3) and 4,5-epoxy-β-caryophyllene (7)

Bioactivity in the BPIB showed three active pools: FII (2.91 g), FIII (3.11 g), and FV (9 g). FII, eluted with hexane/CHCl₃, 7 : 3, was subjected to CC (silica gel, 27.5 g) using hexane/EtOAc, 99 : 1, to yield secondary fractions FII-A-FII-C. According to the BPIB, the activity was concentrated in fraction FII-B. Preparative TLC (silica gel, benzene/CHCl₃, 7 : 3) yielded 7 (22.4 mg). From active fraction FIII, eluted with hexane/CHCl₃, 6 : 4, were obtained 11.9 mg of 2 as a glassy solid, after extensive preparative TLC (silica gel), using benzene/EtOAc, 9 : 1. Active fraction FV (eluted with CHCl₃) was further resolved on a silica gel column (180 g) eluting with a concentration gradient of benzene/EtOAc, starting with benzene, to afford four secondary fractions (FV-A-FV-D). The BPIB indicated that the phytotoxic activity was concentrated in fractions FV-B and FV-C, eluted with benzene/EtOAc, 99 : 1 and 95 : 5, respectively; preparative TLC (silica gel, benzene/EtOAc, 8 : 2) of fraction FV-B yielded 3 (145 mg). From secondary fraction FV-C crystallized 1 (25 mg).

Isolation of 7α,8α-epoxygrindelic acid (4), 7α-hydroxy-8(17)-dehydrogrindelic acid (5) and 17-hydroxygrindelic acid (6) as their methyl esters 4 a, 5 a and 6 a, respectively

Inactive fraction FVI (6.16 g), eluted with CHCl₃/MeOH, 9.9 : 0.1, was methylated with ethereal CH₂N₂. The resulting methylated fraction was further purified by column chromatography on silica gel (60 g) employing mixtures of increasing polarities of benzene and EtOAc to afford three secondary fractions, namely FVI-A-FVI-C. From secondary fraction FVI-B, eluted with benzene/EtOAc, 97 : 3, a solid residue (40 mg) was precipitated which was purified by preparative TLC (silica gel, benzene/EtOAc, 9 : 1) to render 16 mg of the methyl ester 4 a as a yellowish resin. Primary fraction FVII (3.01 g), eluted with CHCl₃/MeOH, 8 : 2, was column chromatographed on silica gel (38 g); the elution process was carried out with CHCl₃/MeOH, 99 : 1, to yield secondary fractions FVII-A-FVII-C. Fraction FVII-B (0.34 g) was methylated with ethereal CH₂N₂. Preparative TLC (silica gel) of the methylated FVII-B using CHCl₃/MeOH, 7 : 3, allowed the isolation of compound 5 as its methyl ester derivative 5 a (33 mg). Finally, primary fraction FVIII (5.6 g), eluted with CHCl₃/MeOH, 8 : 2, was subjected to silica gel (60 g) CC. The column was also eluted with CHCl₃/MeOH, 99 : 1. This process afforded four secondary fractions (FVIII-A-FVIII-D). Fraction FVIII-B (0.29 g) was treated with ethereal CH₂N₂ and then purified by preparative TLC (silica gel), using hexane/CHCl₃, 1 : 1, as the developing solvent, to yield 6 mg of 6 a as a glassy solid.

Physical and spectral properties of 8α,13S-epoxylabdane-14S,15-diol (1)

8α,13S-epoxylabdane-14S,15-diol (1): Colorless crystals; m.p. 65 - 67 °C; [α]_D: + 29 (c, 0.80, CHCl₃); IR ν_max (KBr) cm^-1: 3417, 2938, 2869 and 1099; NMR (see Tables [1] and [2]); FAB⁺-MS (nba): m/z 325 [M + 1 (15)], 307 (45), 245 (100), 191 (35), 149 (55), 81 (45), 69 (58), 43 (55), 41 (46). HREIMS m/z 324.493 (calcd for C₂₀H₃₆O₃, 324.498).

Acetylation of 8α,13S-epoxylabdane-14S,15-diol (1): A solution of 1 (10 mg) in pyridine (0.1 ml) and Ac₂O (0.1 ml) was kept for 48 h at room temperature. Workup as usual afforded the diacetate 1 a, 11 mg, as a glassy solid. IR ν_max (film) cm^-1: 2930, 2869, 1745, 1458, 1242, 1043 and 993; ¹H-NMR (300 MHz, CDCl₃): δ = 5.03 (1H, dd, J = 8.8, 2.6 Hz, H-14), 4.46 (1H, dd, J = 12.0, 2.7 Hz, H-15a), 4.08 (1H, dd, J = 11.8, 8.6 Hz, H-15b), 2.10 (3H, s, OAc), 2.02 (3H, s, OAc), 1.20 (3H, s, H-17), 1.19 (3H, s, H-16), 0.87 (3H, s, H-18), 0.80 (6H, s, H-19 and H-20); EI-MS: m/z = 408 [M⁺ (1)], 263 (99), 245 (100), 189 (9.3), 163 (25.7), 137 (68.6), 123 (29), 81 (25), 69 (25), 43 (53.2).

Isolation of 8α,13S-epoxy-15-nor-labdan-14-al (1 b): To a solution of 1 (20 mg) in EtOH (2 ml) were added 2 ml of 0.1 N ethanolic solution of HIO₄. The mixture was left at room temperature in the dark during 24 h. The reaction mixture was made alkaline with saturated NaHCO₃ and extracted with CHCl₃. The organic phase was dried over anhydrous Na₂SO₄ and evaporated in vacuo to yield 16 mg of 8α,13S-epoxy-15-nor-labdan-14-al (1 b) as an oil. [α]_D: + 42 (c, 1.0, CHCl₃); IR ν_max (KBr) cm^-1: 2932, 2867 and 1765; NMR (see Tables [1] and [2]); HREIMS m/z 292.455 (calcd for C₁₉H₃₂O₂, 292.456).

Isolation of 8α,13S-epoxy-15-nor-labdan-14-oic acid methyl ester (1 d): A solution of 1 b (8 mg) in acetone (2.3 ml) was treated with Jones reagent (100 μl). The mixture was heated under reflux during 2 h. Excess reagent was eliminated by adding EtOH; the resulting mixture was diluted with water and extracted with CHCl₃ (15 ml × 2). Evaporation of the solvent afforded 7 mg of 8α,13S-epoxy-15-nor-labdan-14-oic acid (1 c), which was treated with an excess of an ethereal solution of CH₂N₂ to yield 6.5 mg of 1 d, m.p. 101 °C [Lit. m.p. 99 - 100 °C (4)]; [α]_D: + 30 (c, 1.0, CHCl₃) [Lit. [α]_D: + 30.5 (4)]; IR ν_max (KBr) cm^-1: 2932, 2867, 1755 and 1151; ¹H-NMR (300 MHz, CDCl₃): δ = 3.81 (3H, s, COOMe), 2.42 (1H, m, H-12a), 1.85 (1H, dt, J = 12.0, 3.5 Hz, H-7a), 1.69 (1H, m, H-6a), 1.56 (1H, m, H-1a), 1.15 (3H, s, H-17), 1.07 (3H, s, H-16), 0.86 (3H, s, H-18), 0.78 (3H, s, H-19), 0.70 (3H, s, H-20); HREIMS m/z 322.487 (calcd for C₂₀H₃₄O₃, 322.482).

Isolation of 15-benzoyloxy-8α,13S-epoxylabdane-14S-ol (1 e): To a stirred solution of 1 (7.5 mg) and 1-(benzoyloxy)benzotriazole (5.8 mg), prepared as previously described [5], in CH₂Cl₂ (4 ml) was added triethylamine (3.6 μl). The reaction mixture was stirred at room temperature for 18 h, diluted with CH₂Cl₂ (10 ml), washed with saturated NaHCO₃ solution (10 ml) and brine (10 ml), dried over anhydrous Na₂SO₄, and evaporated to dryness. The resulting mixture was purified by column chromatography on silica gel (10 g). Elution with CH₂Cl₂ yielded 6.5 mg of 1 e as an oily residue. ¹H-NMR (300 MHz, CDCl₃): δ = 8.09 (2H, m, H-2′ and H-6′), 7.56 (1H, m, H-4′), 7.44 (2H, m, H-3′ and H-5′), 4.47 (1H, dd, J = 11.4, 1.5 Hz, H-15a), 4.15 (1H, dd, J = 11.7, 8.1 Hz, H-15b), 3.98 (1H, dt, J = 2.4, 5.4 Hz, H-14), 1.32 (3H, s, H-16), 1.14 (3H, s, H-17), 0.87 (3H, s, H-18) and 0.79 (6H, s, H-19 and H-20); HREIMS m/z 428.613 (calcd for C₂₇H₄₀O₄, 428.604).

Preparation of (S)- and (R)-MTPA ester derivatives 1 f and 1 g: To a solution of 1 e (1.5 mg in 0.8 ml of CDCl₃) were sequentially added pyridine-d ₅ (0.1 ml), 4-(dimethylaminopyridine) (0.5 mg) and (R)-(-)-α-methoxy-α-(trifluoromethyl)-phenylacetyl chloride (25 mg). The mixture was heated at 50 °C for 4 h under nitrogen atmosphere to give the S-Mosher ester 1 f (¹H-NMR data, Table [3]). Treatment of 1 e (1.5 mg) with (S)-(+)-α-methoxy-α-(trifluoromethyl)-phenylacetyl chloride (25 mg) as described above, yielded the R-Mosher ester 1 g (¹H-NMR data, Table [3]).

Physical and spectral properties of methyl grindelate (2)

Methyl grindelate (2): colorless glassy solid; [α]²⁰ _D: - 83 (c, 1.0, CHCl₃); IR ν_max (KBr) cm^-1: 2962, 2920, 1740, 1470, 1443 and 994; NMR (see Tables [1] and [2]); EIMS: m/z 334 [M⁺ (2.5)], 319 (1), 261 (5), 210 (100), 187 (4), 149 (3), 136 (24), 43 (12), 41 (11).

Grindelic acid (3), 7α,8α-epoxygrindelic acid methyl ester (4 a), 7α-hydroxy-8(17)-dehydrogrindelic acid methyl ester (5 a), 17-hydroxygrindelic acid methyl ester (6 a) and 4,5-epoxy-β-caryophyllene (7): Compounds 3 - 6 had been previously isolated from Grindelia pulchella and G. chiloensis and the spectral and physical properties of 3 and the methyl esters 4 a - 6 a were identical to those previously described [6], [7]. The identification of sesquiterpene 7 was performed by comparison with an authentic sample previously isolated from Conyza filaginoides [8].[]

Results and Discussion

The aerial parts of X. gymnospermoides var. eradiatum were extracted with CHCl₃/MeOH, 1 : 1. The initial phytotoxic activity of the resulting extract was evaluated on seedlings of Amaranthus hypochondriacus L. using the Petri dish bioassay [3]. Table [4] summarizes the phytotoxic activity of the extract. The active extract was fractionated by column chromatography over silica gel to yield nine primary fractions (FI-FIX). The bioautographic phytogrowth inhibitory bioassay was used at each step for activity-directed fractionation [3]. Extensive chromatography of the phytotoxic fractions FII, FIII and FV (see Materials and Methods) allowed the isolation of 8α,13S-epoxylabdane-14S,15-diol (1), methyl grindelate (2), grindelic acid (3) and 4,5-epoxy-β-caryophyllene (7). In addition, from the inactive chromatographic pools 7α,8α-epoxygrindelic (4), 7α-hydroxy-8(17)-dehydrogrindelic (5) and 17-hydroxygrindelic (6) acids were obtained as their methyl esters 4 a - 6 a, respectively. Compounds 1 and 2 are new natural products and were identified by spectroscopic and chemical methods. The spectral properties of compounds 3, 4 a - 6 a, including IR and ¹H-NMR data, were identical to those previously described in the literature [6], [7]. The identification of sesquiterpene 7 was performed by comparison with an authentic sample previously isolated from Conyza filaginoides [8].

Compound 1 showed absorptions indicative of hydroxy groups. The HREIMS of 1 gave the M⁺ peak at m/z 324.613 analyzing for C₂₀H₃₆O₃. Upon treatment with Ac₂O/pyridine, 1 afforded the diacetyl derivative 1 a, confirming chemically the presence of two hydroxy functionalities in the molecule. Detailed analysis of the NMR (Tables [1] and [2]) spectra revealed that 1 was a diterpenoid with an 8,13-epoxylabdane skeleton [9], [10]. The spectra showed signals for a hydroxymethylene moiety, a secondary carbinol group, five methyl groups attached to quaternary carbons and, a cyclic ether functionality. The resonances for the methine and methylene group attached to the carbinol groups appeared as an ABX system in the ¹H-NMR spectrum (Table [1]). The corresponding signals were paramagnetically shifted in the ¹H-NMR spectrum of the diacetyl derivative 1 a (see Materials and Methods). An HMBC experiment (Fig. [1]) confirmed the 8,13-epoxylabdane skeleton. The absolute stereochemistry of the labdane oxide skeleton was established by transforming the diterpenoid 1 into 8α,13S-epoxy-15-nor-labdan-14-oic acid methyl ester (1 d) of known absolute configuration [4]. The transformation of 1 into 1 d was accomplished by treating 1 with HIO₄ in EtOH solution to yield 8α,13S-epoxy-15-nor-labdan-14-al (1 b). The aldehyde 1 b has not been previously described in the literature and the NMR data are indicated in Tables [1] and [2]. The NMR spectra of 1 b differed from that of 1 as the signals for the diol moiety were absent. In their place were observed the typical resonances for the aldehyde functionality at δ_H 9.60 (H-14) and δ_C 205.45 (C-14).

Oxidation of aldehyde 1 b with Jones reagent afforded 8α,13S-epoxy-15-nor-labdan-14-oic acid (1 c), which upon methylation with ethereal CH₂N₂ provided the methyl ester 1 d. The spectral properties of 1 d (IR, ¹H-NMR) as well as the [α]_D value were identical with those previously described for 8α,13S-epoxy-15-nor-labdan-14-oic acid methyl ester [4]. The transformations above described define the absolute stereochemistry of the epoxylabdane skeleton as depicted in 1. Thus the carbocyclic skeleton of 1 belongs to the normal series and the methyl groups at C-8 and C-13 are anti. Further evidence of the anti relationship of the methyl groups at C-8 and C-13 in 1, and therefore of an S configuration at C-13, was obtained by cursory inspection of the NOESY spectrum of 1 b which showed the correlations H-14/H-17, H-17/H-20 and H-20/H-19. The correlation H-14/H-17 was consistent with a syn relationship between the aldehyde functionality and the methyl group at C-8 in 1 b, and therefore with an anti relationship between the methyl groups at C-8 and C-13 in compound 1. The absolute configuration at C-14 was established by using the advanced Mosher's methodology [11]. In order to apply this methodology, first the primary hydroxy group of 1 was protected by selective benzoylation with 1-(benzoyloxy)benzotriazole (5) to yield 1 e. Analysis of the ¹H-NMR data (Table [3]) of the (S)- and (R)-MTPA esters derivatives 1 f and 1 g, prepared from 1 e, showed that Δδ_H (S-R) for H-16, H-12, H-15a and H-15b were 0.16, 0.25, - 0.17 and - 0.17, respectively. Therefore, the absolute stereochemistry at C-14 was established as S. Based on the above evidence compound 1 was identified as 8α,13S-epoxylabdane-14S,15-diol. It is important to point out that the ¹H-NMR spectrum of 1 a was identical to that of the diacetyl derivative of ent-8,13β-epoxylabdane-14S,15-diol (8, barbatol), an ent-labdane previously isolated from Sideritis arborescens Salzm. (Labiatae) [10]. Furthermore, the optical rotation sign [α]_D of 1 and barbatol are opposite. These observations suggested that 1 and barbatol (8) could be enantiomers. However, the absolute configuration at C-14 of barbatol was established as S [10] as follows: (-)-13-epimanoyl oxide (9) was treated with osmium tetroxide to yield two epimeric diols in a ratio of 9 : 1 [10]. The minor diol (8) turned out to be identical to barbatol. The major one, 8 a, epimeric to barbatol at C-14, was converted into ent-8,13β-epoxylabdane-14-ol (8 b). Horeau's method [12] of partial resolution applied to 8 b affords (-)-α-phenylbutyric acid defining the 14S absolute configuration. On the other hand, application of Brewster's benzoate rule [13] to 8 b and 8 c also defines as S the absolute stereochemistry of C-14. Finally, the authors concluded that barbatol (8), an epimer at C-14 of compound 8 a, is ent-8,13β-epoxylabdane-14S,15-diol. Without any doubt, the Horeau's method results defined an S stereochemistry at C-14 in the major diol 8 a, but not in barbatol which should have an R configuration at C-14 due to its epimeric relationship with 8 a. Therefore, it is our opinion that although the methodology applied to determine the absolute configuration at C-14 in barbatol was correct, the conclusions were not. The structures 8, 8 b and 8 c are depicted according to the conclusions of the work by von Carstenn-Lichterfelde et al. [10].

Compound 2 has been previously synthesized from compound 3 on treatment with ethereal CH₂N₂ [6], [7]. However, this is the first report of its isolation from a natural source. The spectral properties of 2 were in agreement with those previously described [6], [7].

Natural products 1 - 3 and 7 were evaluated for their ability to inhibit the seed germination and radicle growth of A. hypochondriacus. Table [4] summarizes the effect of the isolates on radicle growth. In general, the tested compounds were less potent as germination inhibitors (IC₅₀ > 10^-3 M in all cases, data not shown) than as growth inhibitors. The activity displayed by compound 1 was comparable with that of 2,4-D, used as a positive control. However, compounds 2 and 3 were more potent than 2,4-D. Previously, as a part of an ecological study of the Florida scrub community, Fischer and coworkers demonstrated that 17-hydroxygrindelic acid (6) and related metabolites present in Chrysoma pauciflosculosa, a dominant shrub on open sites in the Florida panhandle, reduced the germination and radicle growth of two sandhill grasses, Schizachyrium and Leptochloa [14]. However, in the present investigation, the fraction containing acid 6 did not show significant inhibitory activity on radicle growth of A. hypochondriacus suggesting that this species is less sensitive to acid 6. On the other hand, the methyl ester derivative 6 a only inhibited radicle growth of A. hypochondriacus by 65 % at a concentration of 2.85 × 10^-3 M (1000 ppm). The results of the present investigation indicated that X. gymnospermoides var. eradiatum contains potent phytogrowth-inhibitors that could be involved in the allelopathic interactions of the species in its normal habitat.

Acknowledgements

This work was supported by a grant from CONACyT (convenio 27978N). We thank Isabel Chávez, Beatríz Quiroz, Luis Velasco-Ibarra, Javier Pérez-Flores and Rocío Patiño, Instituto de Química, UNAM, for recording some NMR, MS, UV and IR spectra. We are also grateful to Rosa I. del Villar, Oscar S. Yañez-Muñoz, Graciela Chávez, Marisela Gutiérrez and Georgina Duarte-Lisci, Facultad de Química, UNAM, for obtaining some NMR, IR, and mass spectra. The technical assistance of Laura Acevedo, Perla Castañeda and Daniel Chávez is also acknowledged. J. Aguilar, V. Chávez, E. Herrera, D. Martínez, L. Nava, P. Olguin, F. Basurto and M. Trejo provided field assistance. The U.S. Agency for International Development financed the fieldwork. I. Rivero-Cruz acknowledges the fellowship awarded by CONACyT to carry out graduate studies.

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Dr. Rachel Mata

Facultad de Química Universidad Nacional Autónoma de México

Coyoacán 04510

México D.F.

México

Email: rachel@servidor.unam.mx

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Dr. Rachel Mata

Facultad de Química Universidad Nacional Autónoma de México

Coyoacán 04510

México D.F.

México

Email: rachel@servidor.unam.mx