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Original Article

In vitro anti-Trypanosoma cruzi activity of methanolic extract of Bidens pilosa and identification of active compounds by gas chromatography-mass spectrometry analysis

Parasites, Hosts and Diseases 2023;61(4):405-417.
Published online: November 28, 2023

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4Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados de IPN. Av. IPN No. 2508, Col. San Pedro Zacatenco, Del. Gustavo A. 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*Correspondence: (lucio.galavizsl@uanl.edu.mx)
• Received: June 7, 2023   • Accepted: August 14, 2023

© 2023 The Korean Society for Parasitology and Tropical Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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In vitro anti-Trypanosoma cruzi activity of methanolic extract of Bidens pilosa and identification of active compounds by gas chromatography-mass spectrometry analysis
Parasites Hosts Dis. 2023;61(4):405-417.   Published online November 28, 2023
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In vitro anti-Trypanosoma cruzi activity of methanolic extract of Bidens pilosa and identification of active compounds by gas chromatography-mass spectrometry analysis
Parasites Hosts Dis. 2023;61(4):405-417.   Published online November 28, 2023
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In vitro anti-Trypanosoma cruzi activity of methanolic extract of Bidens pilosa and identification of active compounds by gas chromatography-mass spectrometry analysis
Image Image Image
Fig. 1 Representative results of thin layer chromatography (TLC) analysis with visible light at left and UV spectrum (254 nm) at right. Origin is indicated with arrow heads and Rf with 2 arrows. (A) Chromatographs with chloroform eluent show 2 spots (Rf of 0.07 and 0.17). (B) Ethyl acetate eluent no spot was observed. (C) Methanol eluent the Rf was of 0.82 and 0.88. (D) Chloroform-ethyl acetate 1:1 eluent 2 spots were observed (Rf of 0.04 and 0.15). (E) Ethyl acetate-methanol 1:1 eluent and one spot (Rf= 0.88) was observed. (F) Chloroform-methanol 1:1 eluent the chromatograph shows 1 spot (Rf=0.88), all of which were from methanolic B. pilosa extract.
Fig. 2 Chromatogram mass spectra of different compounds obtained from the BPFC3MOH1 fraction with their respective retention time and molecular structure. (A) Chromatogram shows the first 5 retention times of the identified compounds. (B) Chromatogram of the last 3 identified compounds. The y-axis shows the relative abundance of each molecule as a function of their mass-charge ratios. Identification of the compound with the retention time (min) for each peak is presented in Table 1.
Fig. 3 Inhibitory activity (%) of each compound against Trypanosoma cruzi survival. (A) Evaluation of anti-Trypanosoma activity in the presence of crude extract Bp-mOH. (B) Inhibition of fractions in epimastigotes. (C) Inhibition of fractions in trypomastigote stage. The values are shown as the mean of the percent inhibition±SD. Means of NL and B strains are not significantly different based on the Tukey test for multiple comparisons of means (P>0.05). All assays were performed in triplicate.
In vitro anti-Trypanosoma cruzi activity of methanolic extract of Bidens pilosa and identification of active compounds by gas chromatography-mass spectrometry analysis

Identification of the 8 compounds identified by GC-MS of the BPFC3MOH1 fraction

No. Compound (IUPAC name) Synonyms Classification Molecular formula Retention time (min) % totala
1 2-Furanmethanol, 5-ethenyltetrahydro-α,α,5-trimethyl-, cis- Linalool oxide B
Linalool oxide, cis-
Terpene with an alcohol group C10H18O2 2.3 15.4
2 2-Nonynoic acid, ethyl ester Ethyl 2-nonynoate
Ethyl non-2-ynoate
Flavoring agent C11H18O2 2.4 9.3
3 2-Hydroxy-3-methyl benzaldehyde 3-Methylsalicylaldehyde
2,3-Cresotaldehyde
Benzaldehyde C8H8O2 2.6 50.9
4 -3,7-dimethylnona-2,6-dienal Ethyl citral
2,6-Nonadienal, 3,7-dimethyl-
Fragrance component C11H18O 2.8 2.5
5 Pentadecyl prop-2-enoate Pentadecyl acrylate
2-Propenoic acid, pentadecyl ester
Ester C18H34O2 5.2 22.1
6 Benzene-1,4-diol Hydroquinone
Quinol
Phenol C6H6O2 13.6 0.1
7 2-hydroxy-5-methyl benzaldehyde 5-Methylsalicylaldehyde
Benzaldehyde, 2-hydroxy-5-methyl-
Aromatic aldehyde C8H8O2 16.6 0.3
8 Dodecyl prop-2-enoate Dodecyl acrylate Carboxylic ester C15H28O2 21.4 0.1

a% total=total percentage of each compound present in the sample.

Retention times, identification of compounds (IUPAC name), molecular formula, and structure matched with the NIST-MS software.

Lethal concentrations (LC50) and cytotoxic concentration (CC50) obtained from epimastigotes and trypomastigotes of A. salina against Vero cells treated with Bp-mOH extract and their fractions

Extract/fractions NL LC50 (μg/ml) B LC50 (μg/ml) Artemia salina CC50 (μg/ml) Vero cells CC50 (μg/ml) SI
Bp-mOH ES 318±83.2 432±89.8 2,652±19.1 1,003±10.2 3.1
Bp-mOH TS 552±75.5 329±70.8 2,652±19.1 1,003±10.2 3.1
BPFC1CL1 ES 530±52.5 544±54.5 1,493±19 1,128±12 2.1
BPFC2AE1 ES 518±52.7 533±55.8 1,570±19 1,312±12 2.4
BPFC3MOH1 ES 390±62 400±66 1,655±19 1,594±13 4
BPFC1CL1 TS 843±31.8 864±95 1,286±15 1,099±6 1.3
BPFC2AE1 TS 622±12.1 673±50.4 1,199±8 1,209±15 1.9
BPFC3MOH1 TS 504±22.9 519±39.6 1,301±11 1,289±12 2

SI, selectivity index: CC50/LC50; NL, Nuevo León strain; B, Brener strain of T. cruzi; ES, epimastigote stage; TS, trypomastigote stage; Bp-mOH, methanolic extract; BPFC1CL1, chloroformic collective fraction; BPFC2AE1, ethyl acetate collective fraction; BPFC3MOH1, methanolic collective fraction.

Evaluation of the percentage hemolytic activity of the B. pilosa extract and fractionsa

Concentration (μg/ml) Negative controlb Bp-mOH BPFC1CL1 BPFC2AE1 BPFC3MOH1
300 0 0 0±0.1c 0 0
500 0 0 0 0±0.3 0±0.6
650 0 0 0 0.3±0 0.3±0.9
800 0±0.1 0 0 0.62±0 0.6±0.1
950 0±0.2 0.5±0.3 0.4±0.1 1.8±0.2 0.9±0.7
1,100 0±0.1 2.2±0.4 2±0 2.9±0.3 1.25±0.5
1,500 0±0.3 3.3±0.6 3.6±0 4.5±0.1 1.5±0.4
2,000 0±0.1 5.3±0.2 3.9±0.7 5.79±0.1 1.87±0.4
2,500 0±0.2 5.9±0.7 4.8±0.3 8.3±0.6 2.3±1.1

aThe results are given in percentage of hemolysis and were performed in triplicate.

bNegative control, 1×PBS diluent. Positive control=100%: distilled water. Means of treatments are not significantly different based on the ANOVA and Tukey test for multiple comparisons of means (P>0.05).

cThe values are shown as the mean±SD. For abbreviations see Table 2.

Table 1 Identification of the 8 compounds identified by GC-MS of the BPFC3MOH1 fraction

% total=total percentage of each compound present in the sample.

Retention times, identification of compounds (IUPAC name), molecular formula, and structure matched with the NIST-MS software.

Table 2 Lethal concentrations (LC50) and cytotoxic concentration (CC50) obtained from epimastigotes and trypomastigotes of A. salina against Vero cells treated with Bp-mOH extract and their fractions

SI, selectivity index: CC50/LC50; NL, Nuevo León strain; B, Brener strain of T. cruzi; ES, epimastigote stage; TS, trypomastigote stage; Bp-mOH, methanolic extract; BPFC1CL1, chloroformic collective fraction; BPFC2AE1, ethyl acetate collective fraction; BPFC3MOH1, methanolic collective fraction.

Table 3 Evaluation of the percentage hemolytic activity of the B. pilosa extract and fractionsa

The results are given in percentage of hemolysis and were performed in triplicate.

Negative control, 1×PBS diluent. Positive control=100%: distilled water. Means of treatments are not significantly different based on the ANOVA and Tukey test for multiple comparisons of means (P>0.05).

The values are shown as the mean±SD. For abbreviations see Table 2.