Antimutagenic and antioxidant activity of the essential oils of Citrus sinensis and Citrus latifolia

Abstract

The essential oils of Citrus sinensis and Citrus latifolia showed antimycotic activity against Candida spp. isolated from the oral cavity; they are neither mutagenic on the Ames test nor cytotoxic. Their main components are R-(+)-limonene, β-thujene, α-myrcene and γ-terpinene. The aim of this work was to evaluate their antimutagenic and antioxidant capacities. Antimutagenic properties were evaluated against MNNG and ENNG on S. typhimurium TA100; against 2AA on strain TA98 and in front of 4NQO and NOR on strain TA102. Both were antimutagenic against MNNG (p < 0.001) but only C. latifolia was antimutagenic against ENNG (p < 0.001). Both presented antimutagenic activity against 2AA (p < 0.001). They were antioxidant against the ROS-generating compound 4NQO (p < 0.001) and the antibiotic NOR (p < 0.001). In the antioxidant evaluation, the activity in DPPH assay was in a range of 6-23% for C. sinensis and of 22-71% for C. latifolia. Both were antioxidant compared with BHT in β-carotene bleaching assay and were able to decreased apoptosis in HaCat cells stimulated with H₂O₂. The levels of intracellular superoxide ion were lower in the presence of both oils. In conclusion, the essential oils of C. sinensis and C. latifolia are antimutagenic against at least three types of mutagens and have antioxidants properties.

Figure 1

Antimutagenesis in S. typhimurium TA100. (A) Antimutagenesis of Citrus sinensis against mutations induced by MNNG. Spontaneous reversion 161 ± 80 **p < 0.01, ***p < 0.001. (B) Antimutagenesis of Citrus latifolia against mutations induced by MNNG. Spontaneous reversion: 114 ± 10, **p < 0.01, ***p < 0.001. Each point represents the mean of 15 replicas on five independent studies.

Figure 2

Antimutagenesis in S. typhimurium TA100. (A) Antimutagenesis of Citrus sinensis against mutation induced by ENNG. Spontaneous reversion 239 ± 10. (B) Antimutagenesis of Citrus latifolia, against mutation induced by ENNG. Spontaneous reversion: 239 ± 10. Each point represents the mean of 15 replica on five independent studies, *p < 0.05, ***p < 0.001.

Figure 3

Antimutagenesis in S. typhimurium TA98. (A) Antimutagenesis of Citrus sinensis against mutation induced by 2AA. Spontaneous reversion 16 ± 3. ***p < 0.001. (B) Antimutagenesis of Citrus latifolia. Spontaneous reversion 17 ± 5, ***p < 0.001. Each point represents the mean of 15 replicas on five independent studies.

Figure 4

Antimutagenesis in S. typhimurium TA102. (A) Antimutagenesis of Citrus sinensis, against 4-NQO. Spontaneous reversion 266 ± 30 ***p < 0.001. (B) Antimutagenesis of Citrus latifolia, against 4NQO. Spontaneous reversion 266 ± 30 ***p < 0.001. Each point represents the mean of 9 replicas on three independent studies.

Figure 5

Antimutagenesis in S. typhimurium TA102. (A) Antimutagenesis of Citrus sinensis, against NOR. Spontaneous reversion 412 ± 23 ***p < 0.001. (B) Antimutagenesis of Citrus latifolia, with NOR. Spontaneous reversion 412 ± 23 ***p < 0.001. Each point represents the mean of 9 replicas on three independent studies.

Figure 6

Graphic of the % of antioxidation versus concentration of EGCG, C. sinensis and C. latifolia.

Figure 7

Effect of the EOs in the Inhibition of β-carotene bleaching. Control (25 µL Linoleic acid + 0.8 mg β-carotene), BHT (400 mg), C. sinensis (400 mg) and C. latifolia (400 mg). Each point is the mean ± S.D. of three biological experiments performed by triplicate.

Figure 8

Cell apoptosis measured by flow cytometry. (A) Representative density plots in the presence and absence of the EOs. (B) Quantization of early and late apoptosis as % Annexin V-7AAD positive cells. Results are presented as mean ± S.D. ***p < 0.0001.

Figure 9

Inhibition of intracellular superoxide formation. (A) Histogram of the ethidium fluorescence with and without EOs. (B) Quantization of the mean intensity in treated and untreated cells. ***p < 0.0001.