Animals

A total of 24 male Institute of Cancer Research (ICR) mice, aged 5 weeks (weighing 30–34 g), were purchased from the Nomura Siam International Co., Ltd., Bangkok, Thailand. The mice were kept in wood shavings in a standard room with a controlled temperature of 24 ± 1 °C, humidity of 55 ± 10%, under a 12-hour light-dark cycle. All mice were fed with a standard diet and given ultraviolet reverse osmosis water ad libitum. This study was conducted according to the Ethical Principles and Guidelines for the Use of Animals by the National Research Council of Thailand (1999) and the Animals for the Scientific Purpose Act 2015, and was reported in accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. The study protocol was approved by the Animal Care and Use Committee, Faculty of Medicine, Chulalongkorn University (Permission No. 011/2565).

Acetaminophen (Tylenol) was dissolved in distilled water. A 600 mg/kg dose of acetaminophen was prepared and administered orally to mice in APAP groups via intragastric tube. Based on our previous study (unpublished data), the extraction method using water followed by 50% ethanol provided the highest yield of geniposide from the GJE compared to using either water or ethanol alone. Therefore, this extraction method was selected for the current study. Gardenia jasminoides fruits were purchased from a retailed store. The GJE was prepared by first soaking ground, dried Gardenia jasminoides fruits in distilled water for 60 min. The filtrate was then collected, and the crude extract was soaked again in 50% ethanol for 60 min. Filtrates from both steps were mixed and subjected to evaporation using an evaporator (R-300 BUCHI Rotavapor, Switzerland). The content of geniposide in the extract was quantified using High-Performance Liquid Chromatography (HPLC) analysis. Geniposide in the extract was identified using the Liquid Chromatography-Mass Spectrometry method. The peak of geniposide was observed at a retention time of 9.4 min in the HPLC chromatogram (Supplementary Fig. 1). The mass spectra of geniposide at a retention time of 9.4 min showed a mass of 411.13, as illustrated in Supplementary Fig. 2. According to the results, the mass spectrum of gardenia extract at 411 m/z represented the molecular weight of geniposide plus the molecular weight of Na+. These mass spectra were consistent with previous studies that reported MS2 spectra of geniposide at 203, 231, 249, and 379 m/z. The yield of geniposide from the dry extract was 22.67 ± 0.26%. All methods were in accordance with relevant institutional, national, and international guidelines and legislation.

We based the extract dose on the intended dose of geniposide. In the low-dose GJE group, mice were to receive 100 mg/kg/dose of geniposide; therefore, the dose of the extract was 0.44 g/kg/dose (calculated from 100/22.67 * 100/1000). For the high-dose GJE group, using the same calculation method, mice received 0.88 g/kg/dose of GJE extract, which contained 200 mg/kg/dose of geniposide. Both low- and high-dose dry extracts were dissolved in distilled water before being administered to the mice in the treatment groups. The rationale for selecting the doses of geniposide was derived from a study conducted by Yang et al. [18]. In their research, mice were pretreated with geniposide at doses of 10, 30, and 100 mg/kg before inducing hepatotoxicity with APAP. They found that the 100 mg/kg dose was the most effective. In our study, hepatotoxicity was induced with APAP prior to treatment with GJE, which contains geniposide. Therefore, we chose 100 mg/kg of geniposide as a low-dose reference and explored whether 200 mg/kg would exhibit increased effectiveness. According to an acute toxicity study by Tang and colleagues, the LD50 of Gardenia extract was estimated to be more than 15 g/kg/day, and none of the rats that received Gardenia extract at the dose of 15 g/kg/day exhibited any treatment related changes on autopsy [21]. Therefore, the dosage of GJE extract that we used in this study was within the safe range.

Sample size calculation

As malondialdehyde (MDA) was one of the outcomes of interest, we used MDA levels from a study conducted by Yang, et al. [20]. which investigated the protective role of geniposide against acetaminophen hepatotoxicity to calculate the sample size, as shown below:

$$}/} = }}_}/2}}} + }}_}}} \right)}^}}}}^2}}} \over }\limits^ - }_1}-}}\limits^ - }_2}} \right)}^2}}}$$

$$\:\:}^}_}^\:=\frac_-1\right)_^+(_-1)_^}_+\:_-2}$$

n is a sample size, \(\:}_/2}\) = 1.96 (based on Z table with the probability of falsely rejecting a true null hypothesis (α) = 0.05), \(\:}_}\) = 1.645 (based on Z table with the probability of failing to reject a false null hypothesis (\(\:\)) = 0.95), \(\:}^\)= pooled variance, \(}\limits^ - }_1}\)= mean MDA level in the APAP group (12.22 nmol/mg protein), \(}\limits^ - }_2}\) = mean MDA level in APAP + geniposide group (6.39 nmol/mg protein), n1 = sample size in the APAP group [6], \(\:_^\)= standard deviation in APAP group (3.33), \(\:_\) = sample size in APAP + geniposide group [6], \(\:_^\)= standard deviation in APAP + geniposide group (1.67). Using the formula listed above, the calculated sample size was 6 per group.

Experimental protocol

Twenty-four mice were randomly divided into 4 groups (n = 6 per group): (i) Control group, mice were given distilled water, (ii) APAP group, mice received a single dose of 600 mg/kg acetaminophen via intragastric tube, (iii) APAP + low-dose GJE group, mice received a single dose of 600 mg/kg acetaminophen, followed 30 min later by 2 doses of low-dose GJE (0.44 g/kg/dose, containing Gen 100 mg/kg/dose), administered 8 h apart via an intragastric tube, and (iv) APAP + high-dose GJE group, mice received a single dose of 600 mg/kg acetaminophen, followed 30 min later by 2 doses of high-dose GJE (0.88 g/kg/dose, containing Gen 200 mg/kg/dose), administered 8 h apart via an intragastric tube. At 24 h after APAP administration, all mice were euthanized with an injection of overdose sodium pentobarbital intraperitoneally. Blood and liver samples were obtained. Blood samples were used for the measurement of aspartate aminotransferase (AST, Reflotron Plus, Roche Diagnostics, Basel, Switzerland), alanine aminotransferase (ALT, Reflotron Plus, Roche Diagnostics, Basel, Switzerland), interleukin 6 (IL-6, R&D systems, Minneapolis, MN, USA) and tumor necrosis factor-alpha (TNF-α, R&D systems, Minneapolis, MN, USA), according to manufacturer’s manuals.

Hepatic glutathione measurement

Liver tissue samples were homogenized in radioimmunoprecipitation assay buffer (RIPA buffer, pH 7.4) and subsequently centrifuged at 10,000 x g for 15 min at 4 °C. Glutathione (GSH) content was determined using a GSH assay kit (Cayman Chemical Company, Michigan, USA), according to manufacturer’s instructions. Hepatic GSH levels were measured with a microplate reader by detecting the optical density (O.D.) of GSH at 405–414 nm. The results of hepatic GSH were reported as nmol/mg protein.

Hepatic malondialdehyde measurement

Liver tissue samples were homogenized in RIPA buffer (pH 7.4) and subsequently centrifuged at 1,600 x g for 10 min at 4 °C. MDA concentration was quantified using a thiobarbituric acid reactive substance (TBARS) assay kit (Cayman Chemical Company, Michigan, USA), following the manufacturer’s instructions. Hepatic MDA levels were assessed by measuring the O.D. in the range of 530–540 nm. The results for hepatic MDA were expressed as nmol/mg protein.

Liver histopathology

Liver samples were fixed in 10% formaldehyde for 24–48 h and processed using standard techniques before being embedded paraffin blocks. Sections were cut to a thickness of 5 μm and stained with hematoxylin and eosin (H&E). To assess the severity of acetaminophen-induced hepatotoxicity, histopathological changes in the liver were graded according to the grading criteria described by Mark E. Blazka et al. [22]:

Grade 0: Normal histology.

Grade 1: Minimal congestion and necrosis of hepatocytes, limited to the area immediately around the centrilobular vein; many of the lobes are not affected.

Grade 2: Moderate congestion and hemorrhage of the area around the centrilobular vein, extending into the midzonal cells; most lobules are affected. Areas of confluent necrosis are limited to hepatocytes around the centrilobular vein.

Grade 3: Widespread congestion and hemorrhage in the centrilobular and midzonal areas of the liver. Confluent coagulative necrosis involving all hepatocytes in the centrilobular zone; bridging of areas of necrosis between centrilobular zones is common.

Statistical analyses

Comparisons of continuous variables among groups were performed using one-way ANOVA followed by LSD post hoc analysis for normally distributed data and Kruskal-Wallis test with Dunn’s post hoc analysis for non-normally distributed data. The proportion of mice with each pathological grade was compared between groups using Fisher’s exact test. A p-value of less than 0.05 indicated statistical significance. Normally distributed data were presented as mean ± SD, whereas non-normally distributed data were presented as median ± IQR. All comparisons were analyzed using SPSS for MAC version 22.0 (SPSS Inc., Chicago IL, United States).