Protective Effect of Lemon Essential Oil and Its Major Active Component, D-Limonene, on Intestinal Injury and Inflammation of E. coli-Challenged Mice

Abstract

Inflammatory diseases are a major threat to public health. Natural plant essential oils (EOs) possess anti-inflammatory and anti-oxidative activities. The objective of this study was to investigate the anti-inflammatory effect and mode of action of lemon essential oil (LEO), and its main active component, d-limonene, with different doses on intestinal inflammation of mice. Sixty-four 5-week-old male balb/c mice weighing 22.0 ± 1.5 g were randomly assigned into one of 8 treatments (n = 8/treatment), including normal saline group (NS), Escherichia coli (E. coli) group, and either LEO and d-limonene essential oil (DEO) group supplemented at 300, 600, and 1,200 mg/kg of BW, respectively. After the pre-feeding period, the mice were fasted for 12 h, the mice in the NS group and the E. coli group were gavaged with normal saline, and the mice in the LEO group and DEO group were gavaged with respective dose of EOs for 1 week. One hour after the end of gavage on the 7th day, except that the mice in the normal saline group were intraperitoneally injected with normal saline, the mice in the other groups were intraperitoneally injected with the same concentration of E. coli (108 cfu/ml, 0.15 ml per mouse). The antioxidant indexes were measured including superoxide dismutase (SOD), malondialdehyde (MDA), and myeloperoxidase (MPO) in plasma obtained by taking blood from mouse eyeballs. The inflammatory indexes were measured including interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor alpha (TNF-α) in plasma. The tight junction protein indicators were tested include zona occludens 1 protein (ZO-1), occludin and claudin in mouse duodenum. We found that all of the above indexes for E. coli group were different (P< 0.05) with the NS group. The interaction of EO and dose (E × D) were significant (P < 0.01) for all of the indexes. In addition, LEO at 300 mg/kg BW and DEO at 600 mg/kg BW had better antioxidant and anti-inflammation activity on the infected mice, which reduced (P < 0.05) the plasma concentrations of MDA, MPO, TNF-α, IL-1β, and IL-6, but increased (P < 0.01) the concentrations of SOD. Hematoxylin-eosin (H&E) staining of duodenum observation showed that LEO and DEO reduced inflammatory cell infiltration and maintain the orderly arrangement of epithelial cells. Moreover, supplementation of LEO at 600 mg/kg and DEO at 300 mg/kg BW alleviated (P < 0.05) intestinal barrier injury for increasing the relative expression of ZO-1, occludin and claudin mRNA in mice duodenum. These results showed that the pre-treatment with LEO and DEO had protection of intestinal tissue and inflammation in E. coli infected mice. Both LEO and DEO exhibited activity of antioxidant, anti-inflammatory and alleviating intestinal injury, whereas, compared with DEO, LEO can be active at a lower dosage. Furthermore, as the main active component of LEO, the d-limonene appeared to play not only the major role, but also the joint action with other active components of LEO.

Effects of dietary essential oil supplementation on growth performance, carcass yield, meat quality, and intestinal tight junctions of broilers with or without Eimeria challenge

“…Oregano oil supplementation upregulated the mRNA expression of intestinal barrier gene ZO-1 and Claudin-1 in the duodenum and Claudin-1 in the ileum of Eimeria -challenged broilers, which was consistent with the findings from Ruan et al (2021) . The upregulation of tight junction in the epithelial barrier was also reported in colorectal cancer mice model ( Leong et al, 2022 ), female pigs ( Hofmann et al, 2022 ), Escherichia coli -challenged mice ( Zhao et al, 2022 ), and colitis mice ( Raka et al, 2023 ). It is remarkable how essential oil interacts with the monolayer of enterocytes, probably due to their antioxidant capacity ( Toschi et al, 2022 ).…”

Protective Effect of Citrus Medica limonum Essential Oil against Escherichia coli K99-Induced Intestinal Barrier Injury in Mice

“…Kummer et al [ 37 ] have reported that 500 mg/kg D-limonene reduced the TNF-α levels in the peritoneal exudate of mice. Zhao et al [ 20 ] have reported that 600 mg/kg LEO can alleviate the intestinal inflammation and injury induced by E. coli ATCC25922. However, in this study, 300 mg/kg LEO showed enhanced effects on the production of three inflammatory cytokines, which we speculate may be related to the active ingredient and the action dose of citrus EOs.…”

“…However, in this study, 300 mg/kg LEO showed enhanced effects on the production of three inflammatory cytokines, which we speculate may be related to the active ingredient and the action dose of citrus EOs. Some citrus EOs do not have anti-inflammatory effects at low concentrations [ 38 ], and Zhao et al reported that D-limonene, the main active component of LEO, also had a slight promoting effect in terms of E. coli -induced intestinal inflammation in mice at a dosage of 300 mg/kg [ 20 ]. Based on the above results, citrus EOs have an effect in alleviating intestinal inflammation caused by ETEC, which may derive from the dosage of LEO and the excellent anti-inflammatory effect of the chemical components, such as D-limonene and pinene in LEO [ 36 , 37 ].…”

“…Forty Balb/C mice (half male and half female, 6 weeks, 20.0 ± 1.6 g) were randomly divided into 5 groups as follows: the (1) CON group (normal saline + Tween 80), the (2) K99 group (injected E. coli K99), and the (3–5) EOL, M, and H groups (supplemented 300, 600, and 1200 mg/kg BW LEO, respectively, + E. coli K99). The dose of LEO was determined in our previous study [ 20 ], and LEO was dissolved in Tween 80 and subsequently diluted to 300, 600, and 1200 mg/kg with sterile distilled water. Before the test, all of the mice were fasted for 12 h, and then the mice in the CON group and K99 group were given the same amount of normal saline, and the LEO group was given three different doses of LEO via intragastric gavage for 7 consecutive days.…”

“…Studies have shown that LEO has good antibacterial properties against E . coli [ 18 ] and protective effects on the gastric mucosa and barrier function in mice [ 19 , 20 ]. In this study, we aim to further explore the protective effect of LEO on mammalian pathogenic E .…”