| Arecae semen is the ripe seed of Areca catechu L.in Palmae,firstly listed in Yaolu about 1,700 years ago.Its processed products includes raw,fried and coked arecae semen,which are used to treat parasites,dyspepsia,abdominal distension,abdominal pain,diarrhea,edema and jaundice due to the multiple chemical compounds in the arecae semen,such as alkaloids,tannins,fats,flavones,triterpenes and steroids.It has been reported that it has many pharmacological activities including antiparasitic effects,promote digestion,antioxidant effects,anti-depressive effects,antibacterial and antifungal affects and so on.Consequently,the processed products of arecae semen have been widely applied in decoction and Chinese patent medicine.There are 38 prescriptions in the Pharmacopoeia of the People’s Republic of China(2015)containing the processed products of arecae semen.Arecae semen has been widely used in clinical application,but the incidence of adverse reaction of arecae semen was reported low.Few patients might complain of dizziness,abdominal pain,nausea and vomiting,but no serious adverse reaction was reported in the clinical practice.In addition,among ancient TCM monographs,such as Mingyi Bielu,Xinxiu Bencao and Compendium ofMateria Medica,there was no record about the toxicity of arecae semen.Despite of its wide clinical application,arecae semen also caused consternation in the practice owing to the similar chemical compounds(alkaloids and tannins)and its chewed products which were carcinogenic to humans published by the International Agency for Research on Cancer(IARC)in 2004.Hence,the toxicity of the processed products of arecae semen and their related mechanism are necessary to be detailed study.This topic is a part of the task of "Toxic research of Areca catechu L." in the research of"Decreasing toxicity through the compatibility of four kinds of clinical commonly used traditional Chinese medicine ".In this study,we aimed to explore the toxic effect of raw arecae semen and its underlying mechanism with the method of sub-chronic toxicity,metabolomics and acute toxicity in Wistar rats or Zebrafish.Objective:1.To evaluate the sub-chronic toxicity of raw arecae semen by oral administration in Wistar rats.2.To investigate the toxicity of raw arecae semen and its underlying mechanism by serum and urinary metabolomics combined with multivariate statistical analysis.3.To assesst the acute toxicity of the processed products of arecae semen and the alkaloids and tannins of raw arecae semen by LC50 in Zebrafish.Methods:1.A total of 120 Wistar rats were randomly divided into 4 groups(15 males and 15 females per group).The treated groups were given raw arecae semen aqueous extract(ASAE)at the dose of 750,1500 and 4500 mg/kg/day by oral administration respectively,and the control group was received distilled water only.The rats and their consumed feed were weighted every 3 days.The clinical changes and mortality were observed and recorded daily.Hematological parameters,biochemical parameters,organ weights,urinalysis and histopathological examination of all rats were tested at the end of the 30-day treatment period and another 10-day recovery period.2.The male Wistar rats were divided into the control group and treated group(n=6 in each group),which were given by gavage with distill water or ASAE once a day for 30 days,respectively.Serum and urinary samples were collected from all the rats after treatment of 7-day,14-day and 30-day for metabolomics analysis.Moreover,biochemistry analysis and histopathological examination were performed at the end of study.3.The Zebrafish were exposed in 12-well plates,20 for every well,containing 4mL of the aqueous extract of processed products of arecae semen,alkaloids or tannins of raw arecae semen with ladder concentrations,respectively.The the Zebrafish were cultivated in the incubator for 24,then calculated the LC50 according the dead Zebrafish.Results:1.Deaths,weight loss,diarrhea,sluggish action,tremors and body curl up were observed in the 1500 and 4500 mg/kg groups during the study.The relative organ weights of liver and testis in male rats of 4500 mg/kg group were significantly different compared with the control group at the end of the treatment period.As for laboratory parameters,there were no significant differences at the dose of 1500 and 4500 mg/kg groups compared with the control group in the study,except the white blood cell count,aspartate aminotransferase,alanine aminotransferase,albumin,blood urea nitrogen,creatinine,glucose and total cholesterol.In addition,the results of histopathological examination showed no significant difference compared with the control group.2.The phenomenon of diarrhea,less physical activity,tremors and body curl up were observed in the treated group.Additionally,the body weights of treated rats were significantly decreased compared with control rats from the 8th day after oral administration.Except the level of creatinekinase in the treated group significantly increased compared with the control group,there were no differences on biochemistry parameters and histopathological test in the two groups.Combined with the methods of principal component analysis,orthogonal projection to latent structure-discrimination analysisand available databases,the treated and control rats were clearly distinguished from each other and 18 metabolites were identified as the potential biomarkers in the arecae semen treated rats.The identified biomarkers indicated that there were perturbations of the phospholipid metabolism,amino acid metabolism and fat acid metabolism in the treated group.Inaddition,with the same metord,13 metabolites were identified as the potential biomarkers in the urinary which indicated the perturbations of tryptophan catabolism,energy metabolism and the disorder of gut microbiota in the treated rats.3.The LC50 value of raw,fried and coked arecae semen were 29.74 μ g/mL,32.47 μg/mL and 40.03 μg/mL,respectively.The contents of alkaloids in raw,fried and coked arecae semen were 3.24%,2.56%and 2.07%,respectively.The contents of tannins in raw,fried and coked arecae semen were 22.72%,26.68%and 18.28%,respectively.The LC50 value of alkaloids and tannins in raw arecae semen were 136.14 μg/mLand 21.52 μg/mL,respectively.Conclusions:1.The experimental results showed that there was no toxicity of ASAE in 750 mg/kg group,but the death,clinical behavior,body weights and some laboratory parameters were significant differences in the 1500 mg/kg and 4500 mg/kg group compared with the control group.Therefore the ASAE of 750 mg/kg/day was safe in rodents over 30-day administration,but it was toxic by prolonged administration with high dosage of AS AE.2.Arecae semen could cause diarrhea,growth retardation,neurovirulence and certain cardiotoxicity in Wistar rats.That may be associated with the perturbations of phospholipid metabolism,amino acid metabolism,fat acid metabolism,energy metabolism and the disorder of gut microbiota in the treated rats induced by arecae semen aqueous extract.In addition,the approach of UPLC-MS metabolomics combined with multivariate statistical analysis is a useful tool to study the toxicity in TCM.3.The toxicity of the raw areca semen is much stronger than that of fried and coked arecae semen which is coincident with the toxicity-reducing effect of processing.The tannins are the poisonous component in raw arecae semen. |
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