Nettle Leaf Water Extracts for Hepatoprotection: Insights into Bioactivity and Mitochondrial Function

Abstract

This study aimed to evaluate the hepatoprotective effects of nettle (Urtica dioica L.) leaf water extracts on oxygen consumption in the fatty acid oxidation (FAO) pathway using an in vitro fatty liver HepG2 cell model and employing an oxygraphy approach. It also examined the impact of these extracts on HepG2 cell lipid accumulation and viability under oxidative stress. The extracts were obtained via maceration with preservatives or by sonication with/without preservatives. Their chemical composition, including polyphenols, vitamins, and minerals, was analyzed. Bioactivity was confirmed through antioxidant and antiglycation in vitro assays. The extracts contained minerals, water-soluble vitamins, and polyphenols, primarily phenolic acids and rutin. Sonication increased the polyphenol yield, advanced glycation end-product (AGE) inhibition, and total antioxidant capacity compared to maceration. The added preservatives enhanced DPPH scavenging, while SOD-mimicking effects were comparable across extraction methods. In the liver steatosis model, the nettle extracts improved HepG2 cell viability under oxidative stress, reduced lipid accumulation, and enhanced mitochondrial oxygen consumption in the FAO pathway at mitochondria complex I. These findings demonstrate the impact of nettle leaf water extracts on oxygen flux in different oxidative phosphorylation states of the FAO pathway and deepen the understanding of nettle's protective role in hepatic steatosis. The obtained results confirm the hepatoprotective effects of nettles through multiple mechanisms, primarily involving antioxidant activity, modulation of lipid accumulation, and mitochondrial protection.

Keywords: AGE inhibition; antioxidants; hepatoprotection; mitochondria; nettle leaf extracts; oxidative stress; oxygraphy; phytochemicals.

Figure 1

Main phenolic compounds obtained from the nettle leaf water extracts.

Figure 2

(a) Total polyphenol content shown as gallic acid equivalents in mg/mL of nettle extracts. (b) Total antioxidant capacity shown as ascorbic acid equivalents in mg/mL of nettle extracts. Abbreviations: Em+ extract obtained via maceration with preservatives; Es+ extract obtained via sonication with preservatives; Es extract obtained via sonication without preservatives. In (a), * p = 0.0002 vs. Es+ and ** p = 0.0005 vs. Es. In (b), * p = 0.0433 vs. Es+ and ** p = 0.0052 vs. Es, according to a one-way ANOVA with Tukey’s multiple comparison test.

Figure 3

DPPH scavenging effect of ascorbic acid and nettle leaf extracts. Es+ extract obtained via sonication with preservatives; Em+ extract obtained via maceration with preservatives; Es extract obtained via sonication without preservatives. * p ≤ 0.05 vs. DPPH concentration when inhibition is 0, according to a one-way ANOVA with Dunnett’s multiple comparison test. ^# p ≤ 0.05 vs. Es+ and Em+ extracts at corresponding concentrations, according to a one-way ANOVA with Tukey’s multiple comparison test.

Figure 4

Inhibition of superoxide anion release by ascorbic acid and the nettle leaf extracts. Es+ extract obtained via sonication with preservatives; Em+ extract obtained via maceration with preservatives; Es extract obtained via sonication without preservatives. * p ≤ 0.05 vs. superoxide anion concentration when inhibition is 0, according to a one-way ANOVA with Dunnett’s multiple comparison test.

Figure 5

Inhibition of AGE formation by aminoguanidine (AG) and the nettle leaf extracts. Es+ extract obtained via sonication with preservatives; Em+ extract obtained via maceration with preservatives; Es extract obtained via sonication without preservatives. * p ≤ 0.05 vs. AGE concentration when inhibition is 0, according to a one-way ANOVA with Dunnett’s multiple comparison test. ^# p ≤ 0.05 Em+ vs. Es+ and Es extracts at corresponding concentrations, according to a one-way ANOVA with Tukey’s multiple comparison test.

Figure 6

Effects of the nettle leaf extracts on cell viability. ^# p ≤ 0.05 vs. the control (HepG2 cells without the extracts), according to a one-way ANOVA with Dunnett’s multiple comparison test. * p = 0.0298, ** p = 0.0007, *** p < 0.0001 vs. the tBH control, according to a one-way ANOVA with Tukey’s multiple comparison test.

Figure 7

Effects of the nettle leaf extract on lipid accumulation in HepG2 cells. ^# p ≤ 0.05 vs. the control (untreated HepG2 cells), according to a one-way ANOVA with Dunnett’s multiple comparison test. * p < 0.0001 vs. the fatty acid mix (FA) control, according to a one-way ANOVA with Tukey’s multiple comparison test.

Figure 8

Measurement of oxygen consumption flux using high-resolution respirometry. Representative graphs depict fatty acid oxidation (F-OXPHOS), FN-OXPHOS (complex I (CI) activity), FNS-OXPHOS (complex I and complex II (CI and CII) activity), FNS-ET (CI and CII and electron transfer (ET) activity) activity in HepG2 cells treated with a nettle extract and/or fatty acid mixture (0.5 mM, 2:1 mix of oleic acid and palmitic acid, respectively). Data are expressed as a percentage from assessed pmol O₂/(s × 10⁶ cells). Statistical analysis was performed using a one-way ANOVA followed by a post hoc Tukey’s test. Data are presented as means (percentage of control data) ± SDs. * p = 0.0018 vs. the control; ^# p = 0.0108 vs. FA; ^## p < 0.0001 vs. FA. Experiments were repeated 12–14 times.

Figure 9

Extract preparation. Abbreviations: EM+: extract obtained via maceration with preservatives; Es+: extract obtained via sonication with preservatives; Es: extract obtained via sonication without preservatives.