Skip to main content
NCBI home page
BMC Veterinary Research logoLink to BMC Veterinary Research
. 2025 Sep 24;21:545. doi: 10.1186/s12917-025-04981-9

Enoxolone pharmacokinetics, tissue distribution, and residue depletion in largemouth bass (Micropterus salmoides)

Chen Li 1,#, Xinnan Zhou 1,#, Yunshan Qiu 1,#, Lin Luo 1, Hongli Liu 1, Ping Ouyang 2, Yi Geng 2, Defang Chen 1, Xiaoli Huang 1,✉
PMCID: PMC12461970  PMID: 40993708

Abstract

Enoxolone, a hydrolyzed component of Chinese herbal medicine, has shown efficacy against Flavobacterium columnare in largemouth bass (Micropterus salmoides). However, its pharmacokinetics and muscle residue depletion in M. salmoides are not well understood. This study analyzed plasma and tissue samples using High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) after exposure to 16 mg/L enoxolone. Results indicated a bimodal plasma absorption pattern, with the first peak at 2 h and the highest concentration (Cmax = 44.67 mg/L) at 24 h. The plasma AUC0−24 h was 813.40 mg·h/L, and the MRT was 36 h. Significant accumulation occurred in the kidneys (Cmax = 2223.33 mg/kg, AUC0−24 h = 7268.967 mg·h/L) which is more than other organs. But the Tmax (72 h) and MRT (51 h) is the longest. Muscle residues declined to 0.0164 mg/kg by day 11 post-immersion and the withdrawal time is at least 15 d. Enoxolone concentrations in plasma and tissues reached the MIC within 4–8 h. Acute toxicity tests showed a 96 h LC50 of 12.17 mg/L, with no mortality at concentrations ≤ 50 mg/L within 24 h. Based on these findings, a 4 h immersion at 16 mg/L is recommended for effective F. columnare control, followed by an 15 d withdrawal period to meet food safety standards. This study offers crucial data for optimizing enoxolone use in aquaculture.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12917-025-04981-9.

Keywords: Enoxolone, Pharmacokinetics, Muscle residue depletion, Flavobacterium columnare, Micropterus salmoides, HPLC-MS/MS

Introduction

Largemouth bass (Micropterus salmoides), a species belongs to the genus Micropterus in the sunfish family (Centrarchidae), has emerged as a significant species in freshwater aquaculture in China since its introduction from North America in the early 1980 s due to its desirable characteristics, including tender flesh, high protein content, rapid growth rate, short reproductive cycle, and robust tolerance to varying environmental conditions. According to statistical data, the total production of M. salmoides in 2023 was reached an impressive 880,000 tons [1]. However, in recent years, Flavobacterium columnare has been recognized as the causative agent of gill rot, skin ulcers, and hemorrhagic in M. salmoides mainly affects juvenile and adult fish with water temperature 25 ~ 28 ℃ during April to June or September to October every year. M. salmoides reared in some ponds and cages may have a high mortality rate as high as 60% [2–4].

F. columnare is a gram-negative bacterium that belongs to the order Flavobacteriales, family Flavobacteriaceae, and genus Flavobacterium. It is extensively distributed across global freshwater environments, thriving within a specific range of ambient temperatures. It can infect most economically important freshwater aquatic species, including carp [5], catfish [6], eel [7], goldfish [8], perch [9], salmonids [10] and tilapia [11]. Furthermore, the highly contagious and rapid onset of “columnaris Disease”, caused by F. columnare, lead to severe economic losses in aquaculture worldwide. The disease is often characterized by severe clinical manifestations, including gill rot, body ulcers, hemorrhages, lethargy, and anorexia [12–14]. Fortunately, in our previous study, we used molecular docking technology to screen specific drug by using ZINC 15 database and Traditional Chinese Medicine Library database for the TonB protein of F. columnare and enoxolone was finally found to be the finest drug with the lowest MIC and good cost-efficient. It can effectively prevent the formation of biofilm and improve the survival rate of M. salmoides infected by F. columnare [15]. So enoxolone would be a candidate drug in controlling the “columnaris Disease” in M. salmoides.

Enoxolone is a pentacyclic triterpenoid hydrolysis product of licorice [16]. During the metabolism of enoxolone by glucuronidase in plants or by intestinal bacteria after oral administration, glycyrrhizin is hydrolyzed to two pentacyclic triterpenes: 18α-glycyrrhetinic acid and 18β-glycyrrhetinic acid, both of which possess a wide range of antioxidant, anti-inflammatory, and anti-microbial activities [17, 18]. Enoxolone itself also exhibits significant antibacterial activity against several pathogens, including Staphylococcus aureus, Escherichia coli, and Neisseria gonorrhoeae [19–21]. Recent studies further reveal its potent inhibitory effects on F. columnare [15], hereby expanding its antimicrobial spectrum. However, the pharmacokinetics and muscle residue depletion characteristics of enoxolone in M. salmoides remain unknown and warrant further exploration. This study aimed to investigate the pharmacokinetics and muscle residue dynamics of enoxolone in M. salmoides when administered via immersion, providing theoretical guidance for its rational use in aquaculture.

Materials and methods

Experimental fish and breeding management

A total of 500 M. salmoides of were purchased from a fish farm in Chengdu, Sichuan, China. The fish were acclimatized and fed in a fiberglass tank for 2 weeks before experimentation and were full feeding twice a day (9:00 am and 9:00 pm) for 14 d. During the acclimation period, the water was changed twice daily with sufficient aeration (Aeration 24 h in advance) after feeding. The M. salmoides were exposed to a light-dark cycle of 12 h:12 h. The water temperature was 24 °C, and dissolved oxygen was ≥ 6.0 mg/L.

Pharmacokinetic study and muscle residue depletion

Pre-laboratory

The enoxolone was purchased from Shanghai selleck Co., LTD. Its main ingredient is 18β-glycyrrhetinic acid with a purity of 99.99%.

Based on previous research, the MIC of enoxolone against F. columnare is 8 mg/L [15] in vitro. To determine the optimal immersion concentration of enoxolone, preliminary experiments were conducted using concentrations that were twice (16 mg/L) and three times (24 mg/L) the MIC (Table S1). The results found that when the drug concentration is 16 mg/L, the blood drug concentration Cmax is 17.1 mg/L, which has exceeded the MIC and can effectively inhibit bacteria. These investigations revealed that a immersion concentration of twice the MIC (16 mg/L) was optimal. Since F. columnare can infect not only the skin and gills but also spread to internal organs like the heart, liver, and spleen via blood circulation [22–24]. Consequently, a immersion concentration of 16 mg/L was selected for further experimentation.

Drug immersion and sampling

A total of 300 healthy M. salmoides of similar weight (13.64 ± 1.94 g) and length (10.18 ± 0.53 cm) were fasted for 24 h before the experiment and were randomly selected and divided into three tanks with 30 L aerated water and 100 fish of each tank. The 480 mg enoxolone was dissolved with 7.5 ml of Dimethyl Sulfoxide (DMSO) and mixed into the prepared tank water above to achieve a concentration of 16 mg/L of enoxolone. A total of 10 fish were randomly selected from each tank at 1, 2, 4, 8, 12, 24, 48, and 72 h after drug continuou immersion. The fish were anesthetized using an ice-water slurry prepared in a 1:1 ratio (volume/volume) until they displayed complete loss of equilibrium, unresponsiveness to tactile stimuli, and cessation of opercular movement. Then, peripheral blood samples, gill, skin, eyes, intestines, liver, heart, kidney, and spleen tissues were collected separately. Tissues of the same type from the 10 fish were pooled as one sample. Plasma samples were collected by drawing blood from the caudal vein into centrifuge tubes with sodium heparin, then centrifuging at 4 ℃, 4000 rpm for 5 min, and collecting the supernatant. Each tissue sample should weigh more than 100 mg, and the plasma volume should exceed 50 µL. All samples were immediately flash-frozen in liquid nitrogen and stored at −80 °C. Fish before drug immersion were used as blank control.

The surviving fish were returned to normal aerated water after 72 h of drug immersion. Then muscle tissue from the longissimus dorsi near the dorsal fins were sampled on days 1, 2, 3, 5, 7, 9, and 11. All harvested muscle samples were promptly flash-frozen with liquid nitrogen and then stored at −80 ℃ for later use.

All procedures and experiments were reviewed and approved by the Animal Research and Ethics Committees of Sichuan Agricultural University and were performed in accordance with the guidelines of the committee (Approval No. 20190031). All efforts were made to minimize animal suffering and to reduce the number of animals used.

Sample handling

A methanol/acetonitrile solution was prepared by mixing equal volumes of methanol and acetonitrile. An internal standard solution of 100 ng/ml was made by adding 1000 µL of Tolbutamide (200 µg/ml) to 2000 ml of the methanol/acetonitrile solution.

For plasma samples, after vortexing for 10–30 s and centrifuging at 4000 rpm for 30 s at 4 °C, 20 µL of supernatant was pipetted into a 96-well plate. Then, 200 µL of the 100 ng/ml internal standard methanol/acetonitrile solution was added. After vortexing for 5 min and centrifuging at 4000 rpm for 10 min at 4 °C, 100 µL of the supernatant was mixed with 100 µL of water and prepared for HPLC-MS/MS analysis. For the internal standard blank control, 20 µL of blank control plasma was added to 200 µL of internal standard methanol/acetonitrile solution and processed similarly. An additional blank control was prepared by adding 20 µL of blank control plasma to methanol/acetonitrile solution.

For tissue samples, the tissue was homogenized in a 50% methanol-water solution (1:4 ratio) using a homogenizer at 60 Hz for 240 s. Fifty µL of homogenate was pipetted into a 96-well plate, followed by the addition of 500 µL of 100 ng/ml internal standard methanol/acetonitrile solution. After vortexing for 5 min and centrifuging at 4000 rpm for 10 min at 4 °C, 100 µL of the supernatant was mixed with 100 µL of water and prepared for HPLC-MS/MS analysis. The internal standard blank control was prepared by adding 50 µL of blank control tissue homogenate to 500 µL of internal standard methanol/acetonitrile solution, following the same procedure. Another blank control was prepared by adding 50 µL of blank control tissue homogenate to methanol/acetonitrile solution.

Preparation of standard curves

The enoxolone control was accurately weighed and dissolved in an appropriate amount of DMSO to make a stock solution of SS-A at a concentration of 2 mg/ml. Refer to Table S2 to configure the enoxolone working curve QC samples using DMSO. Refer to Table S3 to configure the enoxolone working curve and QC samples using plasma and blank tissue homogenates. To obtain the standard curve equation for enoxolone, plot the ratio of the sample peak area to the internal standard peak area (As/Ais) on the y-axis and the concentration C (ng/ml) on the x-axis. Perform linear regression with a weight coefficient of 1/C2.

HPLC-MS/MS determination conditions

The chromatographic conditions for the analysis were as follows: The liquid chromatography system employed was the Shimadzu LC30AD Liquid Phase System, and the chromatographic column utilized was a Synergi 4 μm Fusion-RP 80 A LC Column, having dimensions of 50 × 2 mm with a 10 µL injection volume, which operated at a maintained column temperature of 40 °C. Its flow rate was 0.8 ml/min and the run time was 2.4 min. The mobile phase consisted of an aqueous phase, which was an aqueous solution containing 0.1% formic acid, and an organic phase, which was acetonitrile. Detailed specifics of these chromatographic conditions are presented in Table S4.

As for the mass spectrometry conditions, the ion source was configured to be a Turbo Spray source, functioning in the positive ion detection mode. The scanning method employed was Multi-Stage Reaction Monitoring (MRM). The detailed parameters of the ion source, along with the ions and ion transition voltages used for quantitative analysis, are outlined in Tables S5 and S6 respectively.

Determination of Withdrawal Time (WDT)

Enoxolone is eliminated from the body following first-order kinetics, obeying the exponential elimination equation C = C0e-kt during the elimination phase. The time (WDT) required for the drug concentration in tissues to drop to the specified maximum residue limit (MRL) can be calculated based on the measured tissue drug concentrations during the elimination phase and the set MRL. The calculation formula is as follows: Inline graphic. In the equation, C0 represents the intercept of the residue elimination logarithmic curve, and k denotes the rate constant of the residue elimination curve [25].

Determination of the LC50 for Enoxolone

In preparation for the experiment, six plastic tanks containing 2 L of aerated water each were set up. A total of 60 M. salmoides of similar weight (7.98 ± 2.15 g) and length (8.81 ± 0.84 cm) were randomly assigned to six treatment groups, with each group consisting of 10 fish. Within these groups, the 20, 40, 60, 80, 100 mg enoxolone was dissolved in 0.5 ml of DMSO respectively, and then added the dissolved solution to 2 L of water to achieve the following concentrations: 0 mg/L (control), 10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L, and 50 mg/L. Throughout the experimental period, the mortality rate of the M. salmoides in each treatment group was meticulously monitored and documented at specific time intervals: 1, 2, 4, 6, 8, 12, 24, 48, 72, and 96 h. The LC50 of enoxolone on M. salmoides for 96 h was calculated by the probabilistic calculation method [26].

Data analysis

All data were expressed as mean ± standard error. The pharmacokinetic model fitting and parameter calculations were analyzed using WinNolin (version 8.2) pharmacokinetic software. The pharmacokinetic parameters included Tmax (time to peak), Cmax (peak concentration), AUC0−24 h (area under the curve at 0–24 h), AUC0−t (area under the curve at 0-t), MRT (mean residence time). Statistical difference was analyzed via SPSS 27.0 software (IBM Corp., Chicago, IL, USA). A significant difference was determined using the one-way ANOVA analysis, and the significant level was P < 0.05. Charts were drawn via Graph Pad Prism (Version 9.5.0) and Adobe Illustrator software (Version 26.0).

Results

Validation of Pharmacokinetic methodology

Under the conditions of this experiment, Fig. 1 depict a steady baseline walking pattern and well-shaped peaks for both plasma and tissue standards without interference from extraneous peaks. The retention times for plasma and tissue standards were found to be 0.79 min (Fig. 1A) and 0.85 min (Fig. 1B), respectively. The regression equations and correlation coefficients for plasma and tissue were obtained by plotting the standard curve with the peak area determined by HPLC as the vertical coordinate (Y) and the concentration of the drug in the sample (X) as the horizontal coordinate. Enoxolone plasma standards showed good correlation in the concentration range of 20–10,000 ng/ml, with the fitted curve equation: y = 0.000189x-0.000714, correlation coefficient R2 = 0.999, and the lowest limit of detection was 20 ng/ml (Fig. 2A). Enoxolone tissue standards showed good correlation in the concentration range of 2-100000 ng/ml, with the fitted curve equation: y = 0.000197x + 3.57e-006, correlation coefficient R2 = 0.995, and the lowest limit of detection was 2 ng/g (Fig. 2B).

Fig. 1.

Fig. 1

Open in a new tab

Standard curve chromatogram. A Plasma standard curve chromatogram. B Tissue standard curve chromatogram

Fig. 2.

Fig. 2

Open in a new tab

standard curve of enoxolone. A standard curve in plasma. B standard curve in tissue

Pharmacokinetics of Enoxolone in the plasma

The plasma concentration-time curves were shown in Fig. 3. The concentration of enoxolone in plasma showed a bimodal peak from 0 to 72 h, with the first peak appearing at 2 h and the second peak at 24 h. The Cmax of 44.667 mg/L was attained at 24 h. The concentration-time data were analyzed with a non-compartment model, and the pharmacokinetic parameters obtained were shown in Table 1 displaying an AUC0−24 h of 813.400 mg·h/L, an AUC0−t of 2665.400 mg·h/L, and an MRT of 36 h. Based on the enoxolone concentrations corresponding to 48 h and 72 h, the half-life of the enoxolone was calculated to be approximately 53.7 h.

Fig. 3.

Fig. 3

Open in a new tab

Concentration-time curves in the plasma of M. salmoides after enoxolone immersion. Shaded part indicates the standard error. Letters denote the significant differences between the different time (P < 0.05) (n = 3)

Table 1.

Non-atrial Pharmacokinetic parameters of Enoxolone in the plasma of M. salmoides

Tissue Tmax (h) Cmax (mg/L) AUC0−24 (mg·h/L) AUC0−t (mg·h/L) MRT (h)
Plasma 24.0 44.667 ± 2.843 813.400 ± 14.264 2665.400 ± 44.669 36.0
Open in a new tab

Tissue distribution of enoxolone

The time-concentration curves of enoxolone within different tissues of M. salmoides were depicted graphically in Fig. 4. Concurrently, the pharmacokinetic parameters pertinent to these tissues were tabulated in Table 2. enoxolone was detected in all tissues, with the kidney showing notably high and consistent accumulative levels. A significant finding was the presence of multiple peak patterns in the concentration profiles of enoxolone in the gills, eyes, skin, spleen, and intestines, indicating a complex and dynamic interaction between the uptake, distribution, and elimination processes of enoxolone in these particular tissues. Enoxolone attained their peak concentrations (Cmax) as follows: 2223.333 mg/kg in the kidney, 113.167 mg/kg in the liver, 35.033 mg/kg in the spleen, 21.15 mg/kg in the gills, 28.4 mg/kg in the eyes, 21.417 mg/kg in the heart, 45.85 mg/kg in the skin, and 15.433 mg/kg in the intestines. The peak times varied significantly among different tissues. The gills reached their peak concentration fastest, at 24 h, while the liver and kidney peaked later, at 72 h. Other tissues, including the eye, skin, heart, and intestines, reached their peak concentrations at 48 h. These suggest that the gills absorb enoxolone most rapidly, followed by the eye, skin, heart, and intestine, while the liver and kidney show the slowest distribution.

Fig. 4.

Fig. 4

Open in a new tab

Concentration-time curves in various tissues of M. salmoides after enoxolone immersion. A Concentration-time curves in eight tissues. A1 Concentration-time curves in seven tissues other than the kidneys. A2 Concentration-time curves of seven tissues within 12 h. Shaded part indicates the standard error. Letters denote the significant differences in different tissues at the same time (P < 0.05) (n = 3)

Table 2.

Non-atrial Pharmacokinetic parameters of enoxolone in M. salmoides tissue

Tissue Tmax (h) Cmax (mg/kg) AUC0−24 (mg·h/L) AUC0−t (mg·h/L) MRT (h)
Gill 24.0 21.150 ± 2.039 379.017 ± 17.940 1224.817 ± 77.930 37.2
Eye 48.0 28.400 ± 5.776 238.388 ± 11.264 1312.788 ± 135.852 42.5
Skin 48.0 21.417 ± 2.651 377.325 ± 19.463 1325.725 ± 14.827 38.1
Heart 48.0 45.850 ± 5.902 484.575 ± 29.937 2430.975 ± 158.755 42.6
Spleen 48.0 35.033 ± 4.910 345.017 ± 56.797 1777.017 ± 204.761 42.8
Intestine 48.0 15.433 ± 5.902 197.038 ± 8.016 884.238 ± 52.709 41.6
Kidney 72.0 2223.333 ± 72.476 7268.967 ± 265.556 79768.967 ± 3001.251 51.0
Liver 72.0 113.167 ± 17.917 1291.517 ± 215.863 4680.117 ± 659.751 42.2
Open in a new tab

Depletion of enoxolone in the muscle

In compliance with the maximum residue limit of 0.01 mg/kg established by the positive list system implemented in Japan since 2006 [27], the intake of residues in muscle tissue was monitored. The elimination kinetics of the drug from the muscle at various time intervals were depicted in Fig. 5. Upon transfer to regular water for further cultivation, the muscle tissue was found to contain a drug content of 0.194 ± 0.0919 mg/kg on the 1 dpi. This was followed by a consistent decline in the drug concentration over the subsequent days until the third day. However, there was a significant increase from the fifth to the seventh day, after which it resumed its downward trend, reaching 0.0164 ± 0.0031 mg/kg on the 11 dpi. Based on the enoxolone residue concentrations on the 1 dpi and the 11dpi, the half-life of the drug was estimated to be approximately 2.8 d.

Fig. 5.

Fig. 5

Open in a new tab

Depletion of enoxolone in the muscle of M. salmoides during drug withdrawal period. Shaded part indicates the standard error. Letters denote the significant differences between the different time (P < 0.05) (n = 3)

Based on the above data, the WDT was calculated. The results indicated that k was 0.2471 d−1 and C0 was 0.2483 mg/kg. Assuming that it takes t dpi after enoxolone cessation for the concentration to drop to the MRL (0.01 mg/kg), calculations show that t is approximately 13 d. Validation showed that the residue concentration on 13 dpi (C13) was about 0.01 mg/kg, and on 14 dpi (C14) it was approximately 0.0078 mg/kg. Using the standard deviation of residuals (SE = 0.0031 mg/kg), the time for 95% Upper Confidence Limit (UCL) [28] to fall below MRL is 15 d. Thus, a withdrawal period of 15 d is recommended to ensure residues in muscle tissues fall below 0.01 mg/kg with 95% statistical confidence.

Acute toxicity of Enoxolone on M. salmoides

The results of the challenge experiment in terms of mortality rate are graphically represented in Fig. 6. No death occurred in the 0 mg/L concentration group. Final mortality at 96 h was 30% for 10 mg/L concentration group, 90% for 20 mg/L concentration group, and 100% for 30 mg/L concentration group, respectively. In addition, mortality at 36 h and 48 h was 100% in the 50 mg/L and 40 mg/L concentration groups, respectively. During the high-dose immersion and challenge process, the M. salmoides moved slowly and its body color turned pale. Finally, the 96 h LC50 of enoxolone for M. salmoides was 12.174 mg/L by calculation. Interestingly, across the five different immersion concentrations, the mortality rate was 0% at 24 h. Therefore, no mortality observed at 4 h immersion.

Fig. 6.

Fig. 6

Open in a new tab

Survival rate of M. salmoides. The red dotted line represents 24 h. (n = 10)

Discussion

Enoxolone is the main active component of glycyrrhiza with a long history of traditional Chinese medicine. It is extracted and isolated from the licorice plant and has been demonstrated to possess protective effects on various systems. Specifically, enoxolone has been shown to have beneficial effects on the liver, digestive system, and nervous system [29, 30]. Since the primary target organ of Flavobacterium columnare is the gills, this experiment administered drugs via immersion. To simulate real-world farming conditions, a single-dose, continuous immersion method was used. This study aims to explore the pharmacokinetics and distribution patterns of enoxolone in M. salmoides under these conditions, providing a theoretical basis for rational drug use.

It can be known from the pharmacokinetic results that the enoxolone concentration in plasma and multiple tissues rapidly increased within 1 h after immersion and reached a peak between 24 and 48 h. Moreover, the MRT in all tissues exceeded 36 h, suggesting that M. salmoides have a good absorption effect for enoxolone. Some tissues absorbed the drug relatively slowly, but all exhibited a sustained therapeutic effect. This finding is consistent with previous research conducted by Wu [31] and Zhang [32], thereby substantiating that during the continuous immersion process, there is sustained drug uptake by the fish body over time. After the drug concentration in plasma reaches its peak, it shows a downward trend. This may be because enoxolone acid is lipophilic [33]. Enoxolone’s lipophilic nature facilitates rapid partitioning from plasma into lipid-rich tissues, leading to significant tissue accumulation and consequent plasma concentration decline. This is consistent with the research findings of Phillips [34], Woo [35], and Rairat [36]. While not the primary focus of this study, potential metabolic transformation could contribute to the observed plasma concentration profile, warranting further investigation. The order of Cmax in different tissues was: Kidney > Liver > Heart > Spleen > Eye > Skin > Gill > Intestine, indicating a broad distribution of the drug. But the Tmax of kidney and liver is 72 h which is less than other organs. Although the liver and kidney showed slower absorption rates compared to other tissues, they exhibited the highest Cmax values. This suggests that while initial uptake is slow, these organs have a strong capacity for drug retention and accumulation over time. The kidneys and liver displayed a pronounced ability for drug accumulation, whereas the intestine showed comparatively weaker absorption. The cytochrome P450 enzyme system in the liver is critical for drug metabolism, serving as the principal metabolic pathway for both the parent drug and its metabolites [37–39]. These factors may explain the observed sustained elevation in drug concentrations within the liver and kidneys in this study. In the pharmacokinetic study of enoxolone in rats, a broader and higher tissue distribution was observed, particularly in the kidney and liver [40], aligning with the findings of this paper. It has been determined that numerous enoxolone receptors are naturally present on the surface of hepatocytes, allowing enoxolone to bind specifically to these cells and exhibit effective hepatic targeting [41]. Additionally, it provides renal [17, 42] and cardiac [43] protection. However, the lipophilicity nature, poor bioavailability, and low aqueous solubility of enoxolone significantly diminish its dermal absorption profile [44, 45], consistent with the findings of this study. Otherwise, the distribution of enoxolone in plasma, spleen, gills, eyes, skin, and intestines shows multiple peaks. The observed bimodal pharmacokinetic profile is notable because it may impact drug efficacy and safety. The secondary peak can lead to unexpected drug re-exposure, potentially affecting dosing regimen design. It is generally considered that the most plausible explanations for bimodal or multimodal phenomena include differential absorption across body surface barriers, enterohepatic circulation, and multi-site absorption [46–48], which aligns with findings reported by Zhao [49] and Cao [50]. This phenomenon indicates that drug treatment often requires administration twice a day to maintain the therapeutic concentration, and due to tissue redistribution, the duration of drug withdrawal is prolonged, thereby increasing the complexity of medication administration. These findings provide a scientific basis for the use of enoxolone in medicated baths within the aquaculture industry and enhance the understanding of its mechanism of action within fish. However, this study did not further explore the bimodal phenomenon. Future research will focus on investigating this phenomenon.

Antimicrobial dosing regimens are optimized using PK/PD parameters. Previous research indicated that enoxolone is a concentration-dependent drug [15], so its evaluation utilized AUC0−24 h/MIC and Cmax/MIC [51]. Studies indicate that a threshold of 25 for the AUC0−24 h/MIC ratio may suffice for less severe bacterial infections, while for more severe infections, an ideal target is 100 or higher [52, 53]. In this study, the AUC0−24 h/MIC ratio for enoxolone PK/PD analysis reached 101.675, suggesting robust antibacterial efficacy against severe F. columnare infections.

Currently, the data on Enoxolone residue risk remain limited. But the “positive list system” implemented in Japan in 2006 sets a limit of 0.01 mg/kg for agricultural chemicals without established MRLs [27]. Calculations based on muscle residue depletion indicate a WDT of 13 d to reach ≤ 0.01 mg/kg, and 14 d to drop below 0.01 mg/kg. To address regulatory safety requirements, we applied a 95% upper confidence limit (UCL) approach, the result shous that the MRL is 15 d. Therefore, the WDT is at least 15 d.

The 96 h acute toxicity test, with five concentrations and 0–24 h exposure, showed 0% mortality. Based on these findings, a 16 mg/L concentration with 4 h immersion is recommended. The pharmacokinetic results showed that a 4 h enoxolone immersion resulted in blood concentration exceeded MIC (8 mg/L) in most tissues, except for eyes, skin, spleen, and intestine. At 8 h, all tissues had exceeded 8 mg/L. Therefore, if 16 mg/kg of enoxolone is used for F. columnare treatment, it is recommended that the treatment duration be 4 h.

However, given the 72 h continuous immersion period in this study, the pharmacokinetics, metabolism, and elimination profile of enoxolone after oral administration are not yet established. The specific tissue safety levels and the potential for sodium retention is still unvalidated. Consequently, examining drug metabolism, residue levels, and suitable oral dosages of enoxolone is a logical subsequent research step for its application in treating F. columnare infections. Further research is needed on the bimodal phenomenon as well. Further research addressing these aspects will enhance the understanding of the suitability and safety of oral enoxolone use in managing these infections.

Conclusion

In summary, the study demonstrates that the absorption of enoxolone by M. salmoides under immersed conditions is characterized by a gradual yet efficacious process, leading to a wide distribution of the compound throughout the organism. Importantly, the drug concentrations in plasma and all examined tissues managed to reach the effective inhibitory concentration threshold by the 4th -hour post-immersion. Based on these findings, it is recommended that when 16 mg/L of enoxolone was used for an immersion period of 4 h at a temperature of 24 ℃, the blood drug concentration could be maintained above the MIC concentration, which could effectively inhibit bacteria. Furthermore, to ensure the safe clearance of the drug and minimize residue, a resting period exceeding 15 d is suggested before considering the fish suitable for human consumption or further use.

Supplementary Information

Supplementary Material 1 (28.2KB, docx)

Acknowledgements

We would like to acknowledge Sino Copley Bio-pharmaceutical Technology (Hefei, China) for high-performance liquid chromatography-tandem mass spectrometry. In addition, we would like to acknowledge “The Fishery Research Institute, Chengdu Academy of Agriculture and Forestry Sciences” for its support of this study.

Authors’ contributions

Chen Li: Conceptualization, Formal analysis, Data curation, Validation, Writing-original draft. Xinnan Zhou: Formal analysis, Data curation. Yunshan Qiu: Formal analysis, Data curation. Lin Luo : Conceptualization, Formal analysis, Data curation. Hongli Liu: Conceptualization, Writing-original draft. Ping Ouyang: Supervision, Resources. Yi Geng: Supervision, Resources. Defang Cheng: Supervision, Resources. Xiaoli Huang: Supervision, Conceptualization, Methodology, Writing-Review & Editing. All authors read and approved the final manuscript.

Funding

This study was funded by the Sichuan Province International Science and Technology Innovation Cooperation/Hong Kong, Macao and Taiwan science and Technology Innovation cooperation project Assignment (No. 2024YFHZ0346).

Data availability

All data generated or analysed during this study are included in this published article and supplementary information files.

Declarations

Ethics approval and consent to participate

All procedures and experiments were reviewed and approved by the Animal Research and Ethics Committees of Sichuan Agricultural University and were performed in accordance with the guidelines of the committee (Approval No. 20190031).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Chen Li, Xinnan Zhou and Yunshan Qiu contributed equally.

References

  • 1.Department Of Agriculture Fisheries Administration. 2024 China Fisheries Statistical Yearbook[M]. Beijing: China Agricultural Press, 2024.
  • 2.Qi Jingwen Z, Yanhong C, Meijia et al. Diagnosis and treatment of mixed infection of rhabdovirus, Flavobacterium columnare and Aeromonas hydrophila in perch[J]. Chin J Anim Husb Veterinary Med, 2024,(08): 219–22.
  • 3.Yanchun X, Zheng C, Lingyun L, et al. Research progress on main diseases of largemouth Bass(Micropterus salmoides)[J]. CHINA Anim HEALTH INSPECTION. 2018;35(9):72–6. [Google Scholar]
  • 4.Deng Guocheng B, Junjie L. Common diseases of largemouth bass in pond culture and its prevention and treatment[J]. Guangdong Agricultural Sci. 2011;38(18):102–3. [Google Scholar]
  • 5.Jia Yongfang L, Zhishuai D, Qiyan, et al. Transcriptome analysis of immune-related gene expression in yellow river carp (Cyprinus Carpio var.) after challenge with Flavobacterium columnare[J]. Microb Pathog. 2021;160:105148. [DOI] [PubMed] [Google Scholar]
  • 6.Wang Bingchao Z, Fangzheng S, Zechao, et al. Molecular characteristics, polymorphism and expression analysis of Mhc Ⅱ in yellow catfish(pelteobagrus fulvidraco) responding to Flavobacterium columnare infection[J]. Fish Shellfish Immunol. 2022;125:90–100. [DOI] [PubMed] [Google Scholar]
  • 7.Ponpukdee Nantawut W, Pradit R, Channarong, et al. Detection and identification of a fish pathogen Flavobacterium columnare using specific monoclonal antibodies[J]. Aquaculture. 2021;545:737231. [Google Scholar]
  • 8.Kumar VD, Gaurav R, Kumar PP, et al. Isolation and characterization of Flavobacterium columnare from freshwater ornamental goldfish Carassius auratus[J]. J Environ Biol. 2015;36(2):433–9. [PubMed] [Google Scholar]
  • 9.Morley NJ, Lewis JW. Consequences of an outbreak of columnaris disease (Flavobacterium columnare) to the helminth fauna of perch (Perca fluviatilis) in the queen Mary reservoir, south-east England. J Helminthol. 2010;84(2):186–92. [DOI] [PubMed] [Google Scholar]
  • 10.Staroscik AM, Nelson DR. The influence of salmon surface mucus on the growth of Flavobacterium columnare. J Fish Dis. 2010;31(1):59–69. [DOI] [PubMed] [Google Scholar]
  • 11.Kitiyodom Sirikorn Y, Teerapong T, Kim D, et al. Immersion vaccination by a Biomimetic-Mucoadhesive nanovaccine induces humoral immune response of red tilapia (Oreochromis sp.) against Flavobacterium columnare Challenge[J]. Vaccines. 2021;9(11):1253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Decostere A. Flavobacterium columnare infections in fish: the agent and its adhesion to the gill tissue.[J]. Verh K Acad Geneeskd Belg. 2002;64(6):421–30. [PubMed] [Google Scholar]
  • 13.Pacha RE, Ordal EJ. Histopathology of experimental columnaris disease in young salmon. J Comp Pathol. 1967;77(4):419–23. [DOI] [PubMed] [Google Scholar]
  • 14.Zhao Zhangchun L, Sixue W, Chen, et al. Bioinformatics characteristics and expression analysis of TLR3 and its adaptor protein TRIF in largemouth bass (Micropterus salmoides) upon Flavobacterium columnare infection[J]. Gene. 2023;872:147450. [DOI] [PubMed] [Google Scholar]
  • 15.Li Minghao C, Baipeng X. Identification of TonB-dependent siderophore receptor inhibitors against flavobacterium columnare using a structure-based high-throughput virtual screening method[J]. Front Microbiol. 2024;15:1392178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Hussain Hidayat A, Iftikhar W, Daijie, et al. Glycyrrhetinic acid: a promising scaffold for the discovery of anticancer agents[J]. Expert Opin Drug Discov. 2021;16(12):1497–516. [DOI] [PubMed] [Google Scholar]
  • 17.Maatooq Galal T, Marzouk Amani M, Gray Alexander I, et al. Bioactive microbial metabolites from glycyrrhetinic acid[J]. Phytochemistry. 2010;71(2–3):262–70. [DOI] [PubMed] [Google Scholar]
  • 18.Rufang Jin J, Liu J, Zhang, et al. Effect of glycyrrhizic acid and its derivative TY501 on the proliferation of murine macrophage RAW264.7[J]. Drug Evaluation Res. 2011;34(04):255–7. [Google Scholar]
  • 19.Zhao Yuanyuan, Su X. Antibacterial activity of 18β-glycyrrhetinic acid against Neisseria gonorrhoeae in vitro. Biochem Biophys Rep. 2023;33: 101427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Kong Yaqiong C, Feng S, Zhi, et al. Bioactive ruthenium(II)-arene complexes containing modified 18β-glycyrrhetinic acid ligands[J]. J Inorg Biochem. 2018;182:194–9. [DOI] [PubMed] [Google Scholar]
  • 21.de Breij A, Karnaoukh TG, Schrumpf J, et al. The licorice pentacyclic triterpenoid component 18beta-glycyrrhetinic acid enhances the activity of antibiotics against strains of methicillin-resistant Staphylococcus aureus[J]. Eur J Clin Microbiol Infect Dis. 2016;35(4):555–62. [DOI] [PubMed] [Google Scholar]
  • 22.Yeh HY, Shoemaker CA, Klesius PH. Sensitive and rapid detection of flavobacterium columnare in channel catfish Ictalurus punctatus by a loop-mediated isothermal amplification method[J]. J Appl Microbiol. 2010;100(5):919–25. [DOI] [PubMed] [Google Scholar]
  • 23.Foscarini Roberto. Induction and development of bacterial gill disease in the eel (Anguilla japonica) experimentally infected with flexibacter columnaris: pathological changes in the gill vascular structure and in cardiac performance. Aquaculture. 1989;78(1):1–20. [Google Scholar]
  • 24.Shi Xieyao J, Wenping H, Shuang, et al. Isolation and identification of pathogen from osteoglossum bicirrhosum with gill rot Disease[J]. J Anhui Agricultural Sci. 2018;46(15):75–8. [Google Scholar]
  • 25.Ding Junren A, Xiaohui W, Kaiyu, et al. Studies on tissue distribution and the elimination regularity of Doxycycline residues in Ictalurus punctatus[J]. Acta Hydrobiol Sin. 2012;36(1):7. [Google Scholar]
  • 26.Li C, Wu M, Wang H. A comparison of three methods for calculating the median lethal concentration. Prog Vet Med. 2012;33(09):89–92. [Google Scholar]
  • 27.Foundation The Japan Food Chemical. The Japanese Positive List System for Agricultural Chemical Residues in Foods (Enforcement on May 29, 2006)[EB/OL]. (2011-09-30). https://www.ffcr.or.jp/en/zanryu/the-japanese-positive/the-japanese-positive-list-system-for-agricultural-chemical-residues-in-foods-enforcement-on-may-29-.html. Accessed 29 Oct 2024.
  • 28.Sun Lei W, Yilin Y, Ni, et al. Overview of European guideline on injection site residues of Food - producing Animals[J]. Chin J Veterinary Drug. 2023;57(1):45–51. [Google Scholar]
  • 29.Fu Yu C, Jun, Li Y-J, et al. Antioxidant and anti-inflammatory activities of six flavonoids separated from licorice[J]. Food Chem. 2013;141(2):1063–71. [DOI] [PubMed] [Google Scholar]
  • 30.Liu Hui W, Jinan Z, Wei, et al. Systems approaches and polypharmacology for drug discovery from herbal medicines: an example using licorice[J]. J Ethnopharmacol. 2013;146(3):773–93. [DOI] [PubMed] [Google Scholar]
  • 31.Zhenqi Wu Z, Qi T, Zhang, et al. Comparison of Establishing severe pneumonia models in mice with different conditions of Mycoplasma pneumoniae Infection[J]. Chin Archives Traditional Chin Med. 2023;41(01):9–12. [Google Scholar]
  • 32.Zhang X, Wang X, Wang L, et al. Effects of artificial simulated cold environment on viral load and lung injury in lung tissue of young rats with respiratory syncytial virus infection[J]. Mod J Integr Traditional Chin Western Med. 2022;31(22):3074–7. [Google Scholar]
  • 33.Yu Tianyi Z, Xiaoqin L, Zhiqiang, et al. Evaluation of pharmacokinetics and mechanism of anti-hepatocellular carcinoma of glycyrrhetinic acid[J]. J Jinggangshan University(Natural Sci Edition). 2021;42(5):28–34. [Google Scholar]
  • 34.Phillips Brianne E, Harms Craig A, Lewbart Gregory A, et al. Population pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin in the green sea urchin (Strongylocentrotus droebachiensis) following intracoelomic and immersion administration. J Zoo Wildl Med. 2016;47(1):175–86. [DOI] [PubMed] [Google Scholar]
  • 35.Woo Soo J. Temperature-dependent pharmacokinetics of Trichlorfon in common carp (Cyprinus carpio L.) after bath immersion therapy. J Vet Pharmacol Ther. 2021;44(5):820–8. [DOI] [PubMed] [Google Scholar]
  • 36.Rairat Tirawat., Kuo Yu Shin., Chang Chao Chia., et al. Bath immersion pharmacokinetics of florfenicol in Nile tilapia (Oreochromis niloticus)[J]. Journal of veterinary pharmacology and therapeutics et al. 2020, 43(6): 520–526. [DOI] [PubMed]
  • 37.Liang Y, Huang L, Qin J, et al. Analysis of the correlation between HCV RNA load and liver fibrosis in HCV-infected patients[J]. Int J Lab Med. 2012;12(33):1499–500. [Google Scholar]
  • 38.Weihua Wang X, Zou J, Chen, et al. Correlative analysis between virus volume in urinary sediment from nephrotic syndrome patients and renal damage[J]. Chin J Nephrol. 2006;22(1):4. [Google Scholar]
  • 39.Kuihua Y, Zhao W, Zhang C. Analysis of the correlation between hepatitis B viral load and liver injury. Shandong Med J. 2008;48(34):60. [Google Scholar]
  • 40.Cao Mengxin Z, Jiawei Y, Jian-Guo, et al. Physiology-Based Pharmacokinetic study on 18β-Glycyrrhetic acid Mono-Glucuronide (GAMG) prior to glycyrrhizin in Rats[J]. Molecules. 2022;27(14):4657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Liang J, Wu Z, Wu G, et al. Mechanism of anti-hepatocellular carcinoma action of glycyrrhizic acid and its progress as a liver-targeting ligand[J]. China Pharm. 2017;28(22):3150–4. [Google Scholar]
  • 42.Jeong Hye Gwang., You Ho Jin., Park Sung Jun., et al. Hepatoprotective effects of 18beta-glycyrrhetinic acid on carbon tetrachloride-induced liver injury: inhibition of cytochrome P450 2E1 expression[J]. Pharmacological research et al. 2002, 46(3): 221–227. [DOI] [PubMed]
  • 43.Pastorino Giulia C, Laura S, Sónia, et al. Liquorice (Glycyrrhiza glabra): A phytochemical and Pharmacological review[J]. Phytother Res. 2018;32(12):2323–39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Hao Jiajia S, Yuming W, Qing, et al. Effect and mechanism of penetration enhancement of organic base and alcohol on glycyrrhetinic acid in vitro[J]. Int J Pharm. 2010;399(1):102–8. [DOI] [PubMed] [Google Scholar]
  • 45.Li Shuang Q, Yuqin Z, Suohui, et al. Enhanced transdermal delivery of 18β-glycyrrhetic acid via elastic vesicles: in vitro and in vivo evaluation[J]. Drug Dev Ind Pharm. 2011;38(7):855–65. [DOI] [PubMed] [Google Scholar]
  • 46.Qin J, Xu W, Ai X et al. Studies on pharmacokinetics of Avermectin in grass carp (Ctenopharyngodon idellus)[J]. Journal of Northwest A & F University (Natural Science Edition), 2012, 40(08): 13–20.
  • 47.Song J, Wang X, Wang W, et al. Pharmacokinetics and withdrawal period of neomycin sulfate in tilapia (GIFT)[J]. J Fish Sci China. 2010;17(06):1358–63. [Google Scholar]
  • 48.Ob S, Bergh, Ervik A. Pharmacokinetics of florfenicol in Cod Gadus Morhua and in vitro antibacterial activity against vibrio anguillarum.[J]. Dis Aquat Organ. 2003;56(2):127–33. [DOI] [PubMed] [Google Scholar]
  • 49.Zhao Liujie. Pharmacokinetics of robenidine hydrochloride and Enrofloxacin in Carassius auratus gibelio[D]. Shanghai: Shanghai Ocean University; 2017. [Google Scholar]
  • 50.Cui yu Cao. Kinetics regularities of Tiamulin fumarate in niletilapia (Oreochromis niloticus) and expressionand distribution of metabolism related Genes[D]. Shanghai: Shanghai Ocean University; 2021. [Google Scholar]
  • 51.Ye Bai. Study on the PK-PD modeling of florfenicol against Streptococcus agalactiae [M]. Shanghai: Shanghai Ocean University; 2016. [Google Scholar]
  • 52.Jiahong Peng. The pharmacokinetics and pharmacodynamics of Cotrimoxazole in Portunus trituberculatus and Sarafloxacin. Eriocheir sinensis[M]. Shanghai: Shanghai Ocean University; 2015. [Google Scholar]
  • 53.Li G, Zhang W, Wu C, et al. Progress in research on drug metabolism in juvenile animals[J]. Chin J New Drugs. 2022;31(4):329–36. [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (28.2KB, docx)

Data Availability Statement

All data generated or analysed during this study are included in this published article and supplementary information files.

Articles from BMC Veterinary Research are provided here courtesy of BMC

RESOURCES