Sub-acute Toxicity of Licorice-Sargassum Extract in Sprague-Dawley Rats: Biochemical, Histopathological, and Pharmacokinetic Studies
The combination of licorice and sargassum has been a subject of interest in traditional pharmacology, particularly due to the “eighteen antagonistic medicaments” in Oriental Pharmacology, which caution against their simultaneous administration. Despite this, the classic recipe “Hai-Zao-Yu-Hu-Tang,” which contains both licorice and sargassum, is still in use today. However, the safety profile of this combination has not been clearly defined, necessitating a comprehensive toxicological study to assess its potential risks.
In this study, a sub-acute toxicity test was conducted to evaluate the safety of licorice-sargassum extract in Sprague-Dawley rats. The primary active components of licorice include liquirtin, isoliquiritin, liquiritigenin, isoliquiritigenin, and glycyrrhizic acid (GL). Glycyrrhizic acid can be metabolized into glycyrrhetinic acid (GA) in vivo. Previous research has indicated that the plasma concentrations of GA in rats increase following the oral administration of laminaria-licorice extract compared to licorice extract alone. To further investigate this, an ultra-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (UPLC-TQ/MS) method was developed to simultaneously determine the six main components of licorice in rat plasma.
The study involved Sprague-Dawley rats divided into seven groups, each receiving different treatments orally twice daily for four weeks. The groups included a control group receiving normal saline, low-dose sargassum extract (2.66 g/kg), low-dose licorice extract (2.42 g/kg), low-dose licorice-sargassum extract (4.37 g/kg), high-dose sargassum extract (5.33 g/kg), high-dose licorice extract (4.83 g/kg), and high-dose licorice-sargassum extract (8.75 g/kg). Blood samples were collected through the abdominal aorta to obtain serum for biochemical analysis. The heart, liver, and kidney were weighed, and organ coefficients were calculated. Histopathological examination was conducted using hematoxylin-eosin staining, and the inflammatory response was specifically examined using CD68 immunohistochemistry staining. The compounds were detected using ACQUITYTM Ultra Performance Liquid Chromatography (UPLC) and Xevo TQ/MS, with data processed using Masslynx4.1 software.
For the single-dose study, 12 Sprague-Dawley rats were divided into two groups to receive either licorice extract (4.83 g/kg) or licorice-sargassum extract (8.75 g/kg) on day 1. Blood samples were collected from the orbital vein at various time points post-dosing, and plasma samples were obtained. From day 3, the same rats continued to receive their original daily dosage for another seven days. For the multi-dose study, blood samples were obtained on day 9 following the same protocol.
The results showed that the organ coefficients of the heart, liver, and kidney were significantly higher in the high-dose licorice-sargassum extract (HLS) group compared to the control group. Additionally, the organ coefficients of the liver in both the high-dose licorice (HL) and low-dose licorice-sargassum (LLS) groups were perceptibly increased compared to the control group. No malformations or color changes were found in other organs, suggesting that the licorice-sargassum extract had a marked impact on the organ coefficients of the heart, liver, and kidney.
Serum biochemical indicators also showed distinct elevations after co-administration of sargassum and licorice. The levels of creatine kinase in the HLS group and those of hydroxybutyrate dehydrogenase, lactate dehydrogenase (LDH), and aspartate aminotransferase (AST) in both the LLS and HLS groups were significantly higher than those in the control group, indicating a toxic effect on the heart. The levels of alanine transaminase in the HLS group and those of AST and alkaline phosphatase in both the LLS and HLS groups were markedly increased compared to the control group, suggesting liver toxicity. The levels of blood urea nitrogen in the LL, HL, and HLS groups and those of creatinine in the HL group were significantly higher than in the control group, implying kidney toxicity. Furthermore, the levels of LDH in the LLS and HLS groups, triglycerides in the HS, LLS, and HLS groups, and glucose (GLU) in the HL and HLS groups were perceptibly higher than in the control group, indicating potential glucose and lipid metabolism disorders.
Histopathological examination revealed severe pathological responses in cardiac tissues in the HLS group, with inflammation being the main feature. The LS group showed medium-grade inflammatory cell infiltration and myocardial degeneration, while the HS group exhibited limited infiltration of inflammatory cells. Both the LLS and HLS groups demonstrated inflammatory cell infiltration and inflammatory exudation at the edge of the heart tissue, with neutrophil granulocyte infiltration and destroyed myocardial cells observed in the HLS group. In the liver, the LL group showed inflammatory cell infiltration around the central vein, while the HL group exhibited a mild inflammatory response and cell edema. The LLS group demonstrated moderate lymphatic infiltration of the bile duct, and the HLS group showed lymphatic infiltration of the bile duct and liver cell degeneration, suggesting cardiogenic liver cirrhosis. In the kidney, the primary pathological change was an inflammatory response, with moderate inflammatory cell infiltration observed in the HS and HL groups. The LLS group showed pyelonephritis and inflammatory infiltration around the renal tubules, while the HLS group exhibited thickening of the wall epithelium, proximal tubules, and renal tubules, along with inflammatory cell infiltration around the renal tubules and pyelonephritis. A significant increase in CD68+ macrophages was observed in the HLS group, indicating enhanced inflammation in cardiac, hepatic, and renal injury.
The UPLC-TQ/MS method was validated for quantitative analysis in pharmacokinetic studies, showing linearity, lower limits of detection and quantification, intra- and inter-day precisions, extraction recovery, matrix effect, repeatability, and stability. In the single-dose study, the area under the curve (AUC), Cmax, and Tmax of liquirtin, isoliquiritin, liquiritigenin, and isoliquiritigenin decreased after the administration of licorice-sargassum extract compared to licorice extract alone. This was consistent with the results from the multi-dose administration experiment. The levels of these compounds in the licorice extract were low, suggesting they may not contribute significantly to the toxicity of the licorice-sargassum complex. Administration of a single dose of licorice-sargassum extract increased the AUC and Cmax of GL and decreased the half-life (T1/2). However, following multiple doses, the AUC of GL was slightly decreased, possibly due to the cumulative addition of GA inhibiting the secondary absorption of GL in vivo. The double-peak phenomenon displayed by GL and GA may be explained by the hepatoenteral circulation of licorice. Both single- and multi-dose administration of licorice-sargassum extract significantly increased the AUC and Cmax of GA.
In summary, the licorice-sargassum extract exhibited toxic effects on the heart, liver, and kidney in Sprague-Dawley rats. This toxicity may result from sargassum promoting the absorption of GA in licorice. Further studies are warranted to provide guidance for the combined use of licorice and sargassum in clinical practice and to reveal the chemical basis of the licorice-sargassum complex.
doi.org/10.1097/CM9.0000000000001716
Was this helpful?
0 / 0