Hepatoprotective Effects of Four Brazilian Savanna Species on Acetaminophen-Induced Hepatotoxicity in HepG2 Cells

Abstract

We investigated four Cerrado plant species, i.e., Cheiloclinium cognatum (Miers) A.C.Sm, Guazuma ulmifolia Lam., Hancornia speciosa Gomes, and Hymenaea stigonocarpa Mart. ex Hayne, against acetaminophen toxicity using an in vitro assay with HepG2 cells. The activity against acetaminophen toxicity was evaluated using different protocols, i.e., pre-treatment, co-treatment, and post-treatment of the cells with acetaminophen and the plant extracts. HepG2 cell viability after treatment with acetaminophen was 39.61 ± 5.59% of viable cells. In the pre-treatment protocol, the extracts could perform protection with viability ranging from 50.02 ± 15.24% to 78.75 ± 5.61%, approaching the positive control silymarin with 75.83 ± 5.52%. In the post-treatment protocol, all extracts and silymarin failed to reverse the acetaminophen damage. In the co-treatment protocol, the extracts showed protection ranging from 50.92 ± 11.14% to 68.50 ± 9.75%, and silymarin showed 77.87 ± 4.26%, demonstrating that the aqueous extracts of the species also do not increase the toxic effect of acetaminophen. This protection observed in cell viability was accompanied by a decrease in ROS. The extracts' hepatoprotection can be related to antioxidant compounds, such as rutin and mangiferin, identified using HPLC-DAD and UPLC-MS/MS. The extracts were shown to protect HepG2 cells against future APAP toxicity and may be candidates for supplements that could be used to prevent liver damage. In the concomitant treatment using the extracts with APAP, it was demonstrated that the extracts do not present a synergistic toxicity effect, with no occurrence of potentiation of toxicity. The extracts showed considerable cytoprotective effects and important antioxidant characteristics.

Keywords: acetaminophen; hepatoprotective activity; hepatotoxicity activity.

Figure 1

Chromatographic profile of the C. cognatum aqueous extract (CCAE) with UPLC/MS/MS technique at wavelength of 354 nm. A: Peak 1 (tR 2.01 min; UV Max 230.0, 295.0). B: Peak 2 (tR 2.70 min; UV Max 258.0, 318.0, 366,0), strongly suggestive of a mangiferin compound. C: Peak 3 (tR 3.12 min; UV Max 230.0, 274.0, 337.0), suggestive of a catechin or epicatechin compound.

Figure 2

Chromatographic profile of the H. speciosa aqueous extract (HEAE) with HPLC/DAD technique at the wavelength of 354 nm. A: peak with retention of 14.5 min identified as chlorogenic acid and its respective UV spectrum (λmax: 326,233; purity: 1.00). B: peak with retention time of 27.6 with similarity to rutin with its respective UV spectrum (λmax: 256,353; purity: 1.00).

Figure 3

Chromatographic profile of the G. ulmifolia aqueous extract (GUAE) with HPLC/DAD technique at wavelength of 354 nm. A: peak detected in the GUAE showing a retention time of 27.547 min (similarity index: 0.9973; purity: 1.00; λmax: 256,354). B: spectrum of the rutin standard used for comparison. C: spectrum of the peak detected in the GUAE.

Figure 4

Chromatographic profile of the G. ulmifolia aqueous extract (GUAE) with UHPLC/DAD/MS/MS technique at wavelength of 354 nm. The peaks identified showed similarity to rutin. A: spectrum of the peak with a retention time of 4.10 min; B: spectrum of the peak with a retention time of 4.36 min; and C: spectrum of the peak with a retention time of 4.49 min.

Figure 5

Chromatographic profile of the H. stigonocarpa aqueous extract (HSAE) with HPLC-DAD technique at wavelength of 354 nm. A: Peak with retention time of 27.6 min and its respective UV spectrum (purity: 1.00; λmax: 255,354).

Figure 6

Chromatographic profile of the H. stigonocarpa aqueous extract (HSAE) with UHPLC/DAD/MS/MS at wavelength of 354 nm. The peaks in 4.53 min and 4.65 min showed similarity to rutin. A: spectrum of peak with a retention time of 4.53 min; and B: spectrum of the peak with a retention time of 4.65 min.

Figure 7

HepG2 cell viability after 24 h of pre-treatment with aqueous extracts and toxicity induction with APAP (15 mM). Cell viability values (%) are expressed as mean and standard deviation (n = 3). The + and − signs correspond to the presence and absence of treatment, respectively. The first column corresponds to the control without any treatment. The second column corresponds to the group exposed to APAP only. The third, fourth and fifth column correspond to the treatment with different concentration extract. The sixth column corresponds to the silymarin treatment after exposed to APAP (positive control). (A): Pre-treatment results for aqueous extract of C. cognatum (CCAE). (B): Pre-treatment results for aqueous extract of G. ulmifolia (GUAE). (C): Pre-treatment results for aqueous extract of H. speciosa (HEAE). (D): Pre-treatment results for aqueous extract of H. stigonocarpa (HSAE). Positive hepatoprotection control corresponds to silymarin at a dose of 50 µg/mL (sixth column in the graph). The results of the viability of the different treatments were compared with the APAP and with silymarin using analysis of variance (ANOVA) with Dunnet’s post-test (*, p ≤ 0.05, significantly different from APAP) (#, p ≤ 0.05, significantly different from silymarin).

Figure 8

HepG2 cell viability after 24 h of induction APAP (15 mM) toxicity and post-treatment with aqueous extracts. Cell viability values (%) are expressed as median and inter-quartile range (n = 3). The + and − signs correspond to the presence and absence of treatment, respectively. The first column corresponds to the control without any treatment. The second column corresponds to the group exposed to APAP only. The third, fourth and fifth column correspond to the treatment with different concentration extract. The sixth column corresponds to the silymarin treatment after exposed to APAP (positive control). (A): Post-treatment results for aqueous extract of C. cognatum (CCAE). (B): Post-treatment results for aqueous extract of G. ulmifolia (GUAE). (C): Post-treatment results for aqueous extract of H. speciosa (HEAE). (D): Post-treatment results for aqueous extract of H. stigonocarpa (HSAE). Positive hepatoprotection control corresponds to silymarin at a dose of 50 µg/mL (sixth column in the graph). The results of the viability of the different treatments were compared with APAP and silymarin using Kruskal–Wallis with Dunn’s post-test (* p ≤ 0.05 significantly different from APAP) (#, p ≤ 0.05, significantly different from silymarin).

Figure 9

HepG2 cell viability after 48 h of co-treatment with aqueous extracts and APAP (15 mM). Cell viability values (%) are expressed as median and inter-quartile range (n = 3). The + and − signs correspond to the presence and absence of treatment, respectively. The first column corresponds to the control without any treatment. The second column corresponds to the group exposed to APAP only. The third, fourth and fifth column correspond to the treatment with different concentration extract. The sixth column corresponds to the silymarin treatment after exposed to APAP (positive control). (A): Co-treatment results for aqueous extract of C. cognatum (CCAE). (B): Co-treatment results for aqueous extract of G. ulmifolia (GUAE). (C): Co-treatment results for aqueous extract of H. speciosa (HEAE). (D): Co-treatment results for aqueous extract of H. stigonocarpa (HSAE). Positive hepatoprotection control corresponds to silymarin at a dose of 50 µg/mL (sixth column in the graph). The results of the viability of the different treatments were compared with APAP and silymarin using Kruskal–Wallis with Dunn’s post-test (* p ≤ 0.05 significantly different from APAP) (#, p ≤ 0.05, significantly different from silymarin).

Figure 10

ROS levels after pre-treatment protocol with the aqueous extracts and APAP (15 mM). The results are expressed as median and inter-quartile range (n = 3). The + and − signs correspond to the presence and absence of treatment, respectively. The first column corresponds to the control without any treatment. The second column corresponds to the group exposed to APAP only. The third, fourth and fifth column correspond to the treatment with different concentration extract. The sixth column corresponds to the silymarin treatment after exposed to APAP (positive control). (A): Pre-treatment results for aqueous extract of C. cognatum (CCAE). (B): Pre-treatment results for aqueous extract of G. ulmifolia (GUAE). (C): Pre-treatment results for aqueous extract of H. speciosa (HEAE). (D): Pre-treatment results for aqueous extract of H. stigonocarpa (HSAE). Positive control corresponds to silymarin at a dose of 50 µg/mL (third column in the graph). The results of the different treatments were compared with APAP and silymarin using Kruskal–Wallis with Dunn’s post-test (* p ≤ 0.05 significantly different from APAP) (#, p ≤ 0.05, significantly different from silymarin).

Figure 11

ROS levels after post-treatment protocol with the aqueous extracts and APAP (15 mM). The results are expressed as median and inter-quartile range (n = 3). The + and − signs correspond to the presence and absence of treatment, respectively. The first column corresponds to the control without any treatment. The second column corresponds to the group exposed to APAP only. The third, fourth and fifth column correspond to the treatment with different concentration extract. The sixth column corresponds to the silymarin treatment after exposed to APAP (positive control). (A): Post-treatment results for aqueous extract of C. cognatum (CCAE). (B): Post-treatment results for aqueous extract of G. ulmifolia (GUAE). (C): Post-treatment results for aqueous extract of H. speciosa (HEAE). (D): Post-treatment results for aqueous extract of H. stigonocarpa (HSAE). Positive control corresponds to silymarin at a dose of 50 µg/mL (third column in the graph). The results of the different treatments were compared with APAP and silymarin using Kruskal–Wallis with Dunn’s post-test (* p ≤ 0.05 significantly different from APAP) (#, p ≤ 0.05, significantly different from silymarin).

Figure 12

ROS levels after co-treatment protocol with the aqueous extracts and APAP (15 mM). The results are expressed as median and inter-quartile range (n = 3). The + and − signs correspond to the presence and absence of treatment, respectively. The first column corresponds to the control without any treatment. The second column corresponds to the group exposed to APAP only. The third, fourth and fifth column correspond to the treatment with different concentration extract. The sixth column corresponds to the silymarin treatment after exposed to APAP (positive control). (A): Co-treatment results for aqueous extract of C. cognatum (CCAE). (B): Co-treatment results for aqueous extract of G. ulmifolia (GUAE). (C): Co-treatment results for aqueous extract of H. speciosa (HEAE). (D): Co-treatment results for aqueous extract of H. stigonocarpa (HSAE). Positive control corresponds to silymarin at a dose of 50 µg/mL (third column in the graph). The results of the different treatments were compared with APAP and silymarin using Kruskal–Wallis with Dunn’s post-test (* p ≤ 0.05 significantly different from APAP) (#, p ≤ 0.05, significantly different from silymarin).