Gardenia jasminoides extract mitigates acetaminophen-induced liver damage in mice

Abstract

Background

Acetaminophen (APAP)-induced hepatotoxicity is a potentially life-threatening condition. Gardenia jasminoides fruit extract (GJE), which contains geniposide (Gen) as its major active constituent, possesses anti-inflammatory and antioxidant properties and may help address the underlying pathogenesis of APAP-induced hepatotoxicity. This study aimed to evaluate the effects of GJE in a mouse model of APAP-induced hepatotoxicity.

Methods

Twenty-four male ICR mice were divided into 4 groups (n = 6/group): [1] Control group, mice were given distilled water; [2] APAP group, mice received a single dose of 600 mg/kg APAP; [3] APAP + low-dose GJE group, mice received APAP followed 30 min later by 2 doses of low-dose GJE (0.44 g/kg/dose, containing Gen 100 mg/kg/dose) 8 h apart; [4] APAP + high-dose GJE group, mice received APAP followed by 2 doses of high-dose GJE (0.88 g/kg/dose, containing Gen 200 mg/kg/dose). All mice were euthanized 24 h after APAP administration. Liver tissue was used for histological examination and to measure glutathione (GSH) and malondialdehyde (MDA) levels. Serum was used to determine levels of ALT and inflammatory cytokines (tumor necrosis factor- α (TNF-α) and interleukin-6 (IL-6)).

Results

Liver histopathology showed moderate to severe hepatic necroinflammation in the APAP group, whereas only mild necroinflammation was observed in both treatment groups. Serum ALT levels were significantly elevated in the APAP group compared to the control group but were significantly reduced after low- and high-dose GJE treatment. Serum TNF- α levels were significantly higher in the APAP group than in the control group and were significantly lower after high-dose GJE treatment (135.5 ± 477.2 vs. 35.5 ± 25.8 vs. 74.7 ± 47.2 vs. 41.4 ± 50.8 pg/mL, respectively). Serum IL-6 followed a similar pattern. Hepatic GSH levels were significantly lower in the APAP group compared to the control group but significantly increased after both low- and high-dose GJE treatment (19.9 ± 4.5 vs. 81.5 ± 12.4 vs. 71.4 ± 7.8 vs. 82.6 ± 6.6 nmol/mg protein, respectively). Conversely, hepatic MDA levels were significantly elevated in the APAP group compared with the control group but significantly decreased after high-dose GJE treatment (108.6 ± 201.5 vs. 40.5 ± 18.0 vs. 40.5 ± 16.8 nmol/mg protein, respectively).

Conclusions

Treatment with G. jasminoides fruit extract can alleviate APAP-induced hepatotoxicity, likely through its anti-inflammatory and antioxidant properties.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12906-024-04676-y.

Background

Acetaminophen, also known as N-acetyl-para-aminophenol or APAP, is a widely used medication to alleviate pain and fever in various conditions. The drug is generally safe at a therapeutic dose; however, intentional and unintentional overdose could cause hepatotoxicity and acute liver failure. Hepatotoxicity from acetaminophen overdose is the most common cause of drug-induced liver injury in the United States [1] with 56,000 emergency department visits, 2600 hospitalizations and 500 deaths each year. This problem also remains significant at a global level as it is the second most common indication for liver transplantation worldwide [2]. A study by Gulmez et al. [3] showed that one-sixth of acute liver failure cases leading to liver transplantation listing was attributed to acetaminophen overdose. Acetaminophen toxicities present in phases, starting from non-specific symptoms, such as nausea and vomiting, to hepatic enzyme elevation, jaundice, and finally hepatic failure. The effects could be fatal and are associated with multiple organ failure [1]. According to Chun et al. [4]. 1–2% of untreated patients with acetaminophen induced hepatotoxicity died from hepatic failure.

Among several antidotes for acetaminophen overdose, N-acetylcysteine has been widely accepted as a main standard treatment. Nonetheless, N-acetylcysteine does come with a risk of adverse reactions and has relative contraindications. Anaphylactoid reactions, such as urticaria, angioedema, hypotension and bronchospasm, have been reported in some cases [5]. N-acetylcysteine also has a drug interaction with nitroglycerine making consumption of the latter a contraindication for its use [6]. Identifying alternative avenues for acetaminophen-induced liver injury is an unmet therapeutic need.

Experimental studies on plant-derived natural compounds have been conducted in search of phytochemicals that have hepatoprotective activity. Previous studies have shown successful use of curcumin and aloe vera in ameliorating acetaminophen-induced hepatitis [7, 8]. Among numerous herb formulations with hepatoprotective effects, Gardenia jasminoides fruit extract (GJE) and its major active constituents, geniposide and genipin [9], have been demonstrated to attenuate liver injury through their anti-inflammatory, antioxidant and anti-apoptotic effects [4, 10–16]. Various uses of GJE to treat hepatic conditions have been shown in prior studies, such as the attenuation of hepatic fibrosis by halting hepatic stellate cell activation [10] via the inhibition of sonic hedgehog signaling [11], the amelioration of D-galactosamine and LPS-induced hepatic failure [12] and the protective effects against hepatic ischemia [13]. The potential mechanisms underlying the protective effects of GJE against APAP induced hepatotoxicity have been shown in prior animal studies. In a cholestasis liver disease mouse model, GJE could alleviate hepatic necrosis and lobular inflammation in a dose-dependent manner through the inhibition of toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling, which is also the important signaling pathway in APAP induced liver injury [17]. Moreover, geniposide derived from Gardenia jasminoides J. Ellis was shown to increase hepatic superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and decrease hepatic malondialdehyde (MDA) levels in mice that received carbon tetrachloride [18]. It is important to note that SOD and GSH-Px are important enzymes in the phase 2 biotransformation that protect hepatocytes against APAP toxicity [19]. Geniposide also decreased inflammatory cytokine levels, such as interleukin (IL)-6, IL-1β, and tumor necrosis factor-alpha (TNF-α) in a mouse model of liver fibrosis [18].

With its antioxidant and anti-inflammatory effects shown in other models of liver diseases, we hypothesized that GJE could potentially be used to alleviate APAP induced hepatotoxicity. This hypothesis was supported by a study by Yang et al.. which showed that pre-treatment with geniposide, a major constituent in GJE, could successfully ameliorate APAP induced hepatoxicity by downregulating CYP2E1 expression and inhibiting TLR4/NF-kB signaling pathway [20]. However, in that study, mice were pretreated with geniposide before inducing hepatotoxicity with APAP, which is not applicable to real-life practice where patients present after the ingestion of APAP. Therefore, in our study, we induced hepatotoxicity with APAP first and then treated with GJE afterward. In addition, the mechanisms of which GJE alleviates APAP induced hepatotoxicity need further elucidation. Consequently, the present study aimed to examine the treatment effects and mechanisms of Gardenia jasminoides fruit extract in mice with APAP induced hepatotoxicity.

Materials and methods

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:

n is a sample size, = 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, = mean MDA level in the APAP group (12.22 nmol/mg protein), = 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).

Results

Effects of acetaminophen and Gardenia jasminoides fruit extract on the liver histopathology

Liver histology in the control group was normal. Most mice in the APAP group showed mild congestion and necrosis of hepatocytes in the centrilobular area, with one mouse exhibiting severe congestion and hemorrhage in the centrilobular and midzonal areas with confluent necrosis. Following treatment with low-dose GJE, the majority of mice had normal liver histology, while two mice exhibited mild to moderate congestion and hemorrhage in the centrilobular area. All mice in the high-dose GJE group had normal liver histology. The mean severity scores were higher in the APAP group compared to the control, low-dose GJE, and high-dose GJE groups (1.33 vs. 0 vs. 0.5 vs. 0, respectively, p < 0.05 for all comparisons, as shown in Fig. 1; Table 1).

Fig. 1.

Representative images of liver histopathology in all groups (H&E staining with 400x magnification). A, Control group showed normal liver histology; B, APAP group showed severe congestion and hemorrhage in the centrilobular and midzonal areas with confluent necrosis (Grade 3); C, APAP + low-dose GJE group showed mild congestion and hemorrhage in the centrilobular area (Grade 1); D, APAP + high-dose GJE group showed normal liver histology. Black arrows indicate congestion and hemorrhage. Green yellows indicate confluent necrosis

Table 1.

Hepatic histopathological scores of mice in each experimental group

Group	n	Hepatic necroinflammation score				Mean score
		0	1	2	3
Control	6	6	0	0	0	0
APAP	6	0	5	0	1	1.33 ± 0.82
APAP + low-dose GJE	6	4	1	1	0	0.5 ± 0.84
APAP + high-dose GJE	6	6	0	0	0	0

Data are presented as number of mice exhibiting each score of hepatic necroinflammation according to criteria by Blazka et al. [22]. The proportion of mice with each pathological grade was compared between groups using Fisher’s exact test (p < 0.001 for overall comparisons)

Effects of acetaminophen and Gardenia jasminoides fruit extract on serum transaminases, serum inflammatory markers and hepatic oxidative stress marker

Serum transaminases

As shown in Fig. 2A, serum AST was significantly elevated in the APAP group compared to that in controls (p < 0.001). Significant reduction of AST levels was found in low-dose GJE (194.1 ± 10.4 U/L) and high-dose GJE (114.7 ± 3.5 U/L) groups when compared to the APAP group (589.1 ± 93.5 U/L, p < 0.001 for all comparisons). Similarly, serum ALT (Fig. 2B) was significantly increased in the APAP group compared to that in controls (p < 0.001). After treatment, serum ALT levels were significantly lower in both low-dose GJE (64.2 ± 1.9 U/L) and high-dose GJE (47.0 ± 3.6 U/L) groups when compared to the APAP group (185.6 ± 36.8 U/L, p < 0.001 for all comparisons).

Fig. 2.

Bar charts demonstrating liver enzymes, inflammatory markers, hepatic GSH, and MDA levels in all groups. A, Serum AST levels; B, Serum ALT levels; C, Serum TNF- α levels; D. Serum IL-6 levels; E, Hepatic GSH levels; F, Hepatic MDA levels; * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001, **** = p-value < 0.0001. Normally distributed data were presented as mean ± SD, whereas non-normally distributed data were presented as median ± IQR

Serum TNF-α

Serum TNF-α concentration was significantly higher in the APAP group (135.5 ± 477.2 pg/mL) compared to that in controls (35.5 ± 25.8 pg/mL, p = 0.001, Fig. 2C). The level of TNF- α was significantly decreased in the high-dose GJE group (41.4 ± 50.8 pg/mL, p = 0.006) but not in the low-dose GJE group (74.7 ± 47.2 pg/mL, p = 0.09) compared to the APAP group.

Serum IL-6

As demonstrated in Fig. 2D, the level of IL-6 was significantly higher in the APAP group (245.7 ± 411.2 pg/mL) than that in the control group (85.2 ± 76.2 pg/mL, p = 0.004). The level of IL-6 was significantly decreased in the high-dose GJE group (120.7 ± 58.8 pg/mL, p = 0.04) but not in the low-dose GJE group (126.4 ± 112.7 pg/mL, p = 0.21) compared to the APAP group.

Hepatic GSH

Hepatic GSH was significantly lower in the APAP group in comparison with the control group (p < 0.001, Fig. 2E). GSH was significantly elevated in low-dose GJE (71.4 ± 7.8 nmol/mg protein) and high-dose GJE (82.6 ± 6.6 nmol/mg protein) groups as compared to that in the APAP group (19.9 ± 4.5 nmol/mg protein, p < 0.001 for all comparisons).

Hepatic MDA

Hepatic MDA was significantly elevated in the APAP group in comparison with the control group (p = 0.02, Fig. 2F). The level of the MDA significantly decreased in the high-dose GJE (40.53 ± 16.8 nmol/mg protein) compared to that in the APAP group (108.6 ± 201.5 nmol/mg protein, p = 0.008). There was a trend toward a reduction in hepatic MDA levels in the low-dose GJE group (42.7 ± 55.4 nmol/mg protein, p = 0.07) when compared to the APAP group.

Discussion

In this study, we investigated the role of Gardenia jasminoides fruit extract on APAP-induced hepatotoxicity in a mouse model. We demonstrated that levels of serum hepatic transaminases and inflammatory markers, including IL-6 and TNF-α, significantly decreased in mice treated with Gardenia jasminoides fruit extract compared to those in the APAP group. Significant improvement in hepatic histology, hepatic GSH levels and MDA levels were also observed in Gardenia jasminoides fruit extract treated mice.

At therapeutic doses, 90% of acetaminophen is metabolized through sulfation and glucuronidation pathways, forming non-toxic metabolites while the remaining 10% of the drug is metabolized by CYP450 enzymes, especially CYP2E1, forming a toxic product called N-acetyl-p-benzoquinone imine (NAPQI), which is further conjugated by glutathione into non-toxic metabolites. APAP overdose exhausts sulfation and glucuronidation and the excess APAP is metabolized by CYP2E1 into NAPQI, resulting in depleted GSH reserves in hepatocytes and covalently bonded protein adduction [1]. Adduct formation causes increased superoxide production which triggers the release of free radicals and subsequent development of mitochondrial oxidative stress. Consequently, mitochondrial stress induces mitochondrial permeability transition (MPT) pore opening, releasing pro-apoptotic proteins such as BAX. This results in the disruption of membrane potential and ATP synthesis, causing DNA fragmentation and hepatocyte necrosis [1, 23, 24]. Ultimately, the activation of pro-inflammatory cytokines ensues as evidenced by the elevated levels of IL-6, IL-8 and TNF-α in APAP hepatotoxicity [25, 26].

The use of Gardenia jasminoides fruit as an anti-inflammatory and antioxidant substance has been widely studied in several liver conditions. Geniposide, a major constituent of the Gardenia jasminoides extract, enhances the activities of GSH peroxidase and decrease MDA levels in liver. Geniposide treatment was found to reduce levels of IL-6, IL-1β, and TNF-α in a mouse model of liver fibrosis. Moreover, geniposide was shown to reduce hepatocyte apoptosis by downregulating BAX expression and cleaving caspase-3 [18]. Previous in vitro and in vivo studies have shown that geniposide attenuates oxidative stress and inflammatory response via the modulation of Nrf2/AMPK/mTOR signaling in non-alcoholic fatty liver disease [12] and increase DNA binding activities of hepatocyte nuclear factor-4α and peroxisome proliferator-activated receptor-α in alcoholic steatosis [27]. Similarly, our study showed that geniposide in the Gardenia jasminoides fruit extract reduced serum IL-6, serum TNF-α and hepatic MDA, and increased hepatic GSH in mice with APAP-induced hepatotoxicity attesting to the anti-inflammatory and antioxidant properties of geniposide.

Our findings were also in accordance with a previous study on the use of geniposide as a prophylactic treatment for acetaminophen hepatotoxicity. A study by Yang et al. [20] showed that pretreatment with geniposide before APAP administration at three different doses of 10, 30 and 100 mg/kg had protective effects against APAP-induced hepatotoxicity in mice, with geniposide at 100 mg/kg having the most obvious effects. In that study, similar patterns of biochemical markers were observed in geniposide-treated mice, such as lower serum AST and ALT levels, suppression of CYP2E1 expression, restoration of decreased GSH, reduced MDA levels, reduction of TNF- α and IL-1βc release, reduction of macrophage and neutrophil infiltration, inhibition of TLR4/NF-kB signaling pathway and attenuation of hepatocyte necrosis, as compared with APAP mice. Additionally, we similarly observed a dose-dependent effect of GJE and geniposide on APAP-induced hepatotoxicity. However, our study differed from theirs in terms of study design. In their study, mice were administered geniposide before the induction of hepatotoxicity by APAP. In contrast, our study induced liver damage in mice first and administered treatment afterward, reflecting a more clinically relevant approach.

Since TNF- α is an important pro-inflammatory cytokine triggered by TLR-4 activation, the inhibition of the TLR4/NF-kB pathway by geniposide [20] might explain the reduction in serum TNF- α levels observed in our study. A previous in vitro study also demonstrated that geniposide could activate the PI3K/Akt/mTOR signaling pathway, leading to decreased production of inflammatory cytokines such as TNF- α and IL-6 [13, 28]. Moreover, the suppression of CYP2E1 expression by geniposide might lead to reduced production of NAPQI, a major toxic metabolite of APAP, thereby ameliorating APAP-induced hepatotoxicity [20]. A previous in vitro study suggested that geniposide enhances the antioxidative capacity of hepatocytes by increasing the nuclear translocation of Nrf2 and upregulating the expression of superoxide dismutase, an important antioxidant enzyme [14]. These mechanisms likely explain the reduction in hepatic MDA levels observed in the high-dose GJE group, which contained geniposide. Despite not being directly studied in APAP-induced hepatotoxicity, Gardenia jasminoides fruit extract has been shown to reduce hepatocellular injury and liver fibrosis in a bile duct ligation model [10]. Another animal study also demonstrated that Gardenia jasminoides fruit extract ameliorated hepatocyte necrosis and liver inflammation in an alpha-naphthylisothiocyanate (ANIT)-induced cholestatic injury through the inhibition of the TLR4/NF-κB pathway [17].

Our study is the first to assess the treatment effects of Gardenia jasminoides fruit extract in APAP-induced hepatotoxicity. We found that Gardenia jasminoides fruit extract attenuated acetaminophen induced liver injury via the reduction of hepatic necroinflammation and oxidative stress and the restoration of GSH. However, our study was not without limitations. As we used Gardenia jasminoides fruit extract instead of purified geniposide in our study, we could not say with absolute certainty that other constituents in Gardenia jasminoides fruit extract did not play a role in its treatment effects. Nevertheless, the high-performance liquid chromatography analysis of Gardenia jasminoides fruit extract in this study showed that other substances constituted only a small proportion of the extract. Therefore, we hypothesized that geniposide was likely a main player in the action of Gardenia jasminoides fruit extract. Moreover, we did not use N-acetyl cysteine (NAC) as a positive control; therefore, we were unable to determine whether Gardenia jasminoides fruit extract had effects comparable to the standard of care, such as NAC. However, this could be the focus of a future study.

In conclusion, our findings suggested that Gardenia jasminoides fruit extract, through its inflammatory and antioxidant properties, could potentially be used as a novel therapy for APAP-mediated hepatotoxicity. Further studies are needed to confirm the effects of Gardenia jasminoides fruit extract in clinical settings.

Electronic supplementary material

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Abbreviations

ALT

Alanine aminotransferase

APAP

Acetaminophen

AST

Aspartate aminotransferase

GJ

Gardenia jasminoides

Gen

Geniposide

GSH

Glutathione

IL-6

Interleukin 6

MDA

Malondialdehyde

NAC

N-acetyl cysteine

TNF-α

Tumor necrosis factor-alpha

Author contributions

PT: conducting the experiments, acquisition of data, analysis and interpretation of data, and drafting of the manuscript. MC: study concept and design, analysis and interpretation of data, a critical review of the manuscript and finalizing the manuscript; PW: acquisition of data and conducting the experiments; KS: acquisition of data and conducting the experiments; PS: study concept and design, a critical review of the manuscript; NK: acquisition of data and conducting the experiments; DW: study concept and design, a critical review of the manuscript, and finalizing the manuscript. All authors have read and approved the manuscript for submission.

Funding

This study was funded by the the Grant of Ratchadaphiseksomphot, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (Grant number GA66/074), Center of Excellence in Alternative and Complementary Medicine for Gastrointestinal and Liver Diseases, Department of Physiology, Faculty of Medicine, Chulalongkorn University and Pink Diamond Project, Faculty of Medicine, Chulalongkorn University. Funders provided monetary support but had no role in the study design and experiment, data analysis and interpretation, and manuscript writing.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information file.

Declarations

Ethics approval and consent to participate

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 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 (The permission No. 011/2565).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.