The Active Component Of Xianglian Pill: Extraction,preliminary Safety Evaluation And Effects On 5-Fu Induced Diarrhea Mice

BackgroundXianglian Pill（XLP）is originally documented in"Taiping Huimin Heji Prescription",which is a popular recipe for the treatment of diarrhea.It is widely used clinically for gastrointestinal diseases such as damp-heat dysentery.The XLP recorded in the 2015 edition of the Chinese Pharmacopoeia is in the form of herb powder of Yuhuanglian and Radix Aucklandiae at the ratio of 4:1.The inevitable sequence of herb powder is the large size in dosage which often results in poor patient’s compliance.Thus,concentrated XLP and Xianglian Slice are developed to reduce the dose size.In fact,the dose size of forms of XLP is still complained by patents and their extraction techniques need to be further progressed.Meanwhile,the toxicity profile of XLP is less studied.In addition,the effects of XLP on chemotherapy induced diarrhea still remain unclear.In the current work,we optimized the extraction process of Yuhuanglian and conducted the safety evaluation of the active component of XLP.Moreover,the effect of the active component of XLP on 5-FU induced diarrhea in mice was investigated.Objective:To optimize the extraction process of Yuhuanglian,the main herb drug in the formula of XLP and conduct the safety evaluation of active component of XLP was carried out.To explore the improvement effect of the active components of XLP on chemotherapy induced diarrhea and gut microbiota.Methods:1.Optimization of extraction process of YuhuanglianBased on the results of single factor experiment,taking ethanol concentration,material to liquid ratio,extraction time and extraction temperature at different levels as the influencing factors,the total alkaloid as quality control index,Box-Behnken response surface method was used to design and optimize the extraction process.2.Preparation and content determination of the active components of XLPThe cornelian berberine and the woody extract are mixed according to the composition ratio of XLP（4:1）and lyophilized to obtain the active components of XLP（Among them,the extract of Yuhuanglian is extracted according to the best process,and the Muxiang extract is provided by another student in the research group）.The content of epiberberine,coptisine,palmatine,berberine,costunolide and dehydrocostus lactone in the active components of XLP were determined.The mobile phase is acetonitrile（A）-0.05 mol·L^-1 potassium dihydrogen phosphate solution（B）,gradient elution（0-15 min,30%A;15-40 min,70%A）,the detection wavelength is 225 nm.3.Acute oral toxicity test of the active components of XLP in miceA total of 70 Kunming mice,half male and female,weighing 20±2 g,were randomly divided into 7 groups according to the results of the pre-experiment:normal control group（equivalent volume of saline）and the active components of XLP groups(2.00,2.66,3.54,4.71,6.26 and 8.32 g·kg^-1).Mice in active component group intragastrically administered the active component of XLP at designed dose within 24hours while the mice in control group recived the same volum of water similarly.All mice were observed for 14 days,and death number in each group was recorded.The dead mice were immediately dissected to observe the main organs.Gross morphology and tissue morphology changes were investigated,and finally calculate the organ coefficient and median lethal dose(LD₅₀).4.Chronic toxicity test of the active components of XLP in ratsA total of 80 SD rats in both genders,weighing 200±20 g,were randomly divided into 4 groups:normal control group（equivalent volume of saline）,active component groups of XLP(0.4,0.8 and 1.6 g·kg^-1).Rats in the active component of XLP groups were orally given the active component of XLP for 12 weeks and recovered for 2 weeks while mice in control group were given saline similarly.Changes in appearance,behavior,weight,food and water consumption of the rats during the administration period and recovery period were recorded.After the end of the experiment,rats were anesthetized and sacrificed.Blood was token from the abdominal aorta.Main organs were token out and weighted.The morphological and histopahtological changes were performed.The organ indices were also calculated.Meanwhile blood routine and biochemical indicators were analysised.5.Therapeutic effect of the active components of XLP on 5-FU induced diarrhea in miceChemotherapy diarrhea mouse model was induced by intraperitoneal injection of5-FU at 50 mg·kg^-1 for 5 days.Diarrhea scores,body weight,food and water intake were investigated very day.Generally,mice with diarrhea score more than 1 were considered as diarrhea.After successful modeling,diarrhea mice were divided into a model group,a loperamide hydrochloride capsule(LHC,0.6 mg·kg^-1)treatment group,and the ctive components of XLP(0.1,0.2 and 0.4 g·kg^-1)treatmentgroup.Normal mice were served as normal control group.Mice in treatment groups were orally given corresponding drugs at designed dose once daily for 7 days.The mice in the normal group and the model group were gavaged with an equal volume of saline in the same way.Manifestations of mice during the experiment were recorded.Morphology and pathology of colon,inflammatory cytokines（TNF-α,IL-6 and IL-1β）,oxidative indicators（NO,SOD,GSH and MDA）,short chain fat acids in faeces as well as gut microbiota were detected.Results1.The optimal extraction process of YuhuanglianThe optimal extraction process of the total alkaloids of Yuhuanglian is:ethanol volume fraction 70%,Solid-liquid ratio 1:11(g·ml^-1),extracting twice at 82℃for 2 h each time.The total alkaloid yield was 11.19%,and the deviation from the predicted value of 11.23%was only 0.04%.The total alkaloid content in the homemade active components of XLP is 33.2%2.The content of each ingredient in the active components of XLPThe percentage contents of epiberberine,coptisine,palmatine,berberine,costunolide and dehydrocostus lactone in the active components of XLP determined by HPLC were 4.29%,4.99%,4.14%,18.32%,0.59%and 0.87%,respectively.The total content of main active constituents was calculated as 33.2%.3.Results of acute oral toxicity of the active components of XLP（1）The Dn and Dm value:The Dm of the active components of XLP is 8.2 g·kg^-1and Dn is 2 g·kg^-1.（2）The LD₅₀ value:The LD₅₀ of the active components of XLP is3.712 g·kg^-1（referred to the dried extract）.The 95%confidence interval is 3.145g·kg^-1≤LD₅₀≤4.338 g·kg^-1.Mice in the 8.32 g·kg^-1 group were all died in 24 h after adimistration while mice in control group and in 2.0 g·kg^-1 group were all alive.The long index of dead mice was significantly（P<0.01）higher than that of normal mice.Histopathological investigation revealed the existence of alveolar consolidation,local pulmonary hemorrhage and inflammatory cells in lung cavity in the lung tissues of dead mice.Unclear hepatic cord and inflammatory cell infiltration were also found in the dead mice.The data indicated that the death of mice was largely due to the damages of lung and liver.4.Results of the chronic toxicity of the active components of XLPNo animal death was observed at the end of the experiment.Compared with the normal group,rats in the active components of XLP group(0.4,0.8 and 1.6 g·kg^-1)showed no significant difference（P>0.05）in body weight,food and water consumption,behavoir during the whole experiment period.The organ indices of dosed rats were also normal.Biochemical analysis indicated that PLT,HCB,HCT and Lymph%increased,Mon%and Gran%decreased,and there were no observe abnormalities in other blood routine and biochemical indicators of the active components of XLP treated rats.Histopathological examination revealed that most of the treated rats were normal and some of the rats in high dose group showed liver and lung damage as unclear lung cavity,consolidation,local inflammatory cell infiltration,congestion of the alveolar blood vessels,necrosis of hepatocyte,edema of hepatocyte in the peripheral part,and inflammatory cells infiltration in hepatic lobules were observed from their histopathological pictures.5.Results of therapeutic effect of the active components of XLP on 5-FU induced diarrhea.Intraperitoneal injection of 5-FU at 50 mg·kg^-1 for 5 days,mice developed mild to severe diarrhea judged from DS,body weight,fecal morphologies and spontaneous activities.The active components of XLP significantly alleviated 5-FU-induced body weight loss,diarrhea,decreased thymus and spleen indexes,and pathological changes in the colon.In addition,the active components of XLP remarkably regulated colitis cytokines（TNF-α,IL-6 and IL-1β）and oxidative stress indicators（NO,SOD,GSH and MDA）to normal levels and effectively restored the level of SCFAs.Gut microbiota analysis revealed that the active components of XLP reversed the overall structure of gut microbiota to the normal status.Additionally,the active components of XLP modulated the abundances of different phyla nearly to the normal values and restored the ratios of Firmicutes/Bacteroidetes（F/B）.Conclusions1.The Box-Behnken response surface method can more accurately optimize the extraction process of Yuhuanglian as evidenced by much higher alkaloids the transfer rate of total alkaloids.The extraction largely reduces the dose volume of XLP without the loss of active components of Yuhuanglian.2.The active component of XLP is low-toxicity and long term high dose adiminstraiont of it has the risk of liver and lung damage.3.Although the detailed mechanisms are still remained to be further defined,the active component of XLP are proved to be effective to treat the intestinal toxicities of5-FU.