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Preventive effect of flavonoid extract from peel of Gonggan | JIR

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Introductions

Chemical liver injury is caused by alcohol, chemical toxic substances, drugs, and other hepatotoxic substances. It is a precursor of liver disease, and further development could cause different degrees of hepatocyte necrosis, fatty degeneration, hepatocirrhosis, liver cancer, and other serious liver diseases. It is one of the most common types of liver injury in clinical practice.1,2 Acute liver injury has the characteristics of high morbidity and mortality, which is increasing in recent years.3 Current studies indicated that the pathogenesis of liver injury mainly involves oxidative stress, mitochondrial damage, inflammation, immune regulation, and cell apoptosis, etc.4–6 The occurrence of liver injury usually involves the participation of multiple mechanisms. Therefore, it is of great significance to study the prevention and treatment of acute liver injury.

Flavonoids are common components of natural products and have various physiological activities.7 Natural flavonoids have become important effective components of anti-chemical liver injury. Studies have shown that total flavonoid extracts and monomeric compounds have good anti-chemical liver injury activity.8–10 The mechanisms are related to the inhibition of oxidative stress, inflammation, hepatocyte apoptosis, and other methods of exerting hepatoprotective effects. Citrus flavonoids are one of the main sources of dietary flavonoids and have many biological activities. Studies about the effects of citrus on liver injury are concentrated on the monomer of citrus flavonoids and citrus extract. The extracts of Citrus aurantium L., Citrus depressa, Citrus aurantium, natsumikan (Citrus natsudaidai) and shekwasha (Citrus depressa) have certain protective effect on liver injury, and the liver injury model is established by acetaminophen, alcohol, or D-galactose.11–15 Nobiletin, as a monomer of citrus flavonoids, had protective effect on lipopolysaccharide/D‑galactosamine‑induced liver injury.16 Naringin and hesperidin could inhibit liver injury induced by CCl4.17 The CCl4-induced liver injury is a typical liver injury model, and its pathophysiology is like the pathogenesis of human liver.18 The research on the effect of citrus extract on liver damage caused by CCl4 is few. Citrus peel, a waste product of citrus consumption and processing, is a more convenient source compared with the monomer. Therefore, the study of functional components from citrus peel on liver injury induced by CCl4 could can not only enrich related research but also make full use of the by-products of citrus processing and improve the added value of citrus products.19

In this study, the mouse model of acute liver injury induced by CCl4 was used. The pathological sections, serum, and liver biochemical indicators were analyzed, and the q-PCR results of related genes were explored to evaluate the protective effect and possible mechanism of gonggan peel flavonoid extract (GPFE) on acute liver injury in mice. This study would provide the experimental basis for the development of citrus peel and the research of protecting chemical liver injury.

Materials and Methods

Preparation of GPFE

The peel of gonggan (Citrus reticulata Blanco var. gonggan from Yulin City, Guangxi Zhuang Autonomous Region, China) was freeze-dried and ground into a powder. A solution of citrus peel powder (100 mg) and 80% ethanol solution with a liquid-to-material ratio of 20:1 was heated at 80°C for 4 h.20,21 To purify the crude extract, AB-8 macroreticular resin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was used with a 90% ethanol eluent. When the eluent became colorless, the separation was stopped and the eluates were combined, and evaporated under reduced pressure. The residue was freeze-dried and then ground into powder to be used as the source of GPFE. The total flavonoids of GPFE were obtained by detecting the absorbance at the wavelength of 500nm (Evolution 300 ultraviolet spectrophotometer, Thermo Fisher Scientific, Inc., Waltham, MA, USA) with rutin as standard substance.

Determination of GPFE Composition

Two milligrams each of narirutin, hesperidin, nobiletin, tangeretin, and 5-demethylnobiletin (Shanghai Yuanye Biological Technology Co., Ltd., China) were dissolved in methanol (2 mL, HPLC grade) separately to afford the standard solutions.

A liquid chromatography system (UltiMate3000 HPLC System, Thermo Fisher Scientific, Waltham, MA, USA) with a Welch C18 column (4.6 × 250 mm long, 5 μm) was employed. The mobile phase A was acetonitrile (HPLC grade) and mobile phase B was 0.5% glacial acetic acid aqueous solution. The mobile phase gradient was: 0 min, 12% A; 0~20 min, 25% A; 20~35 min, 45% A; 35~40 min, 100% A. The flow rate was 1.0 mL/min and column temperature was 35°C. After 10 μL test solution was injected, the chromatographic peak area was obtained at detection wavelength of 285 nm.22 The compounds in GPFE were analyzed according the chromatographic peak area of standard substance.

Animal Models and Treatment

Fifty specific pathogen-free (SPF) Kunming mice aged six weeks (20 ± 2g, male) were housed at room temperature 22± 2°C, 55± 5% humidity with 12 h light–dark cycle conditions. After one week of adaptive feeding, the mice were randomly divided into normal group, model group, silymarin group, low concentration GPFE extract group (GPFE-L group), and high concentration GPFE extract group (GPFE-H group). All mice were free to eat and drink. At the same time, the mice in silymarin group were given intragastric administration of 200 mg/kg silymarin; the mice in GPFE-L group and GPFE-H group were given intragastric administration of 150 mg/kg and 300 mg/kg GPFE separately. After 2 weeks, all mice, except the normal group, were peritoneally injected with 0.8% CCl4/olive oil mixture and the induction dose was 10 mL/kg.23,24 After fasting for 16 hours, the blood was taken from the orbit and quickly put at 4°C for 0.5 h, followed by centrifugated at 4000 rpm/min for 10 min.23,24 After the mice were sacrificed by severed neck, the liver was removed immediately and washed in normal saline. The serum and liver were stored at −80°C for further testing. Additionally, the formula: Liver index (%) = organ weight (g)/body weight of mice (g) ×100% was used to calculate the liver index.23

Histological Analysis of the Liver Tissues

The liver tissues (~0.5 cm2) were fixed in 10% formalin solution for 48 h and then embedded in paraffin. The tissues were sectioned and stained with hematoxylin and eosin to observe the pathological changes of liver tissue in each group by an optical microscope (BX43, Olympus, Tokyo, Japan).

Determination of SOD, CAT, and MDA Levels in Liver Tissues

Liver tissue (100 mg) was ground in 900 μL saline solution in ice bath, and then the homogenate was centrifugated at 10,000 rpm for 15 min at 4°C to obtain the test supernatant. The SOD, CAT, and MDA levels in liver tissue were detected by using the kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China).

The method of determining SOD is as follows. According to the kit instruction, the distilled water (20 μL) in blank experiment and supernatant of tissue homogenate (20 μL) in test experiment were added with the enzyme working solution (20 μL) or enzyme diluent (20 μL) as required. All samples were added with applied solution (200 μL). After mixing, the solution was incubated at 37°C for 20 minutes, and the absorbance was measured at 450 nm with Varioskan LUX Multimode Microplate Reader Fluoroskan (Thermo Fisher Scientific, Waltham, MA, USA).

The method of determining MDA is as follows. The reagents provided in the kit were used to prepare reagent 1, reagent 2, reagent 3, and standard solution. Ethanol (0.1 mL), standard solution (0.1 mL) and supernatant of tissue homogenate (0.1 mL) were added separately to different centrifuge tubes, followed by the addition of reagent 1(0.1 mL), reagent 2 (3 mL), and…

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