A high-performance liquid chromatography method was developed for determination of five nucleotides in Bulbus Fritillariae. The five nucleotides were uridine, adenine, guanosine, thymidine, adenosine, respectively. A Welch materials XB-C18 column (4.6 mm x 250 mm, 5 microm) was used and the chromatographic separation was achieved using 5 mmoL x L(-1) ammonium acetate-acetic acid buffer solution (pH 4.30, B) and methanol (A) as mobile phases, the gradient elution program: 0-10 min, 0%-1% A, 10-20 min, 1%-5% A, 20-25 min, 5% A, 25-35 min, 5%-30% A, 35-37 min, 30%-0% A, 37-40 min, 0% A with a flow rate of 1 mL x min(-1) and monitored at 260 nm, the injection volume was 20 microL. The peak areas of nucleotides and the concentrations showed a good linear relation ranged from 0.24 to 13.60 mg x L(-1), r > 0.9983. The intra- and inter-day pecision results were adequate with the RSDs of 2.1% or below. The repeatability was good and the RSD were smaller than 5.5%. The recoveries of nucleosides were in the range of 93.55% and 101.9%, RSD < 3.0%; The order of nucleotides contents in different Bulbus Fritillariae was F. hupehensis > F. thunberqii > F. cirrhosa approximately F. ussuriensis. The method is simple, convenient and accurate. It can be used for the determination of nucleosides and supplying evidence for exploiting and applying of Bulbus Fritillariae.
Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Fritillaria ; chemistry ; Nucleotides ; chemistry ; Reproducibility of Results

ObjectiveTo explore the effect of Qingre Huayu Jianpi prescription （QHJ） on colitis-associated colorectal cancer （CAC） in mice， and its related mechanism. MethodC57BL/6 mice were randomly divided into four groups including the normal， model， QHJ low-dose （QHJ-L， 10 g·kg^-1）， and QHJ high-dose （QHJ-H， 40 g·kg^-1） groups. Azoxymethane （AOM） and dextran sodium sulfate （DSS） were combined to chemically build a CAC mouse model for 14 weeks. Each drug group was given intragastrically from the 5^th week to the 14^th week， once per day. An equal volume of water was fed to the normal and model groups. The mouse survival rate， colon length， weight， and pathological alterations were assessed. The protein expressions of Wnt-3a protein signaling （Wnt3a）， β-catenin， Non-phosphor-β-catenin （Non-p-β-catenin）， and cholesterol-binding glycoproteins 133 （CD133） were detected by Western blot. The localization and expression of the cluster of differentiation （CD） 80 and CD11 antigen-like family member B （CD11b） were detected by immunohistochemistry （IHC）. The colon organoids derived from CAC mice were isolated and cultured to detect the expression of Wnt signaling pathway-related proteins. ResultThe survival rate of the CAC mice was improved by QHJ treatment and the number of colon tumors was inhibited significantly. Compared with those in the normal group， the expression levels of Wnt3a， β-catenin， Non-p-β-catenin， and CD133 in colon tissues in the model group were significantly increased （P<0.05， P<0.01）. Compared with those in the model group， the levels of Wnt3a and β-catenin in the QHJ-L group were significantly decreased （P<0.01）， and the protein levels of Wnt3a， β-catenin， Non-p-β-catenin， and CD133 in the QHJ-H group were significantly decreased （P<0.05， P<0.01）. Meanwhile， the expression level of CD11b in the model group was significantly increased compared with that in the normal group while the CD80 level was significantly decreased （P<0.05， P<0.01）. Compared with those in the model group， CD11b in QHJ-L and QHJ-H groups was significantly decreased， and CD80 was significantly increased（P<0.05， P<0.01）. The expressions of Non-p-β-catenin and CD133 in colonic organoids of CAC model mice were significantly increased， while QHJ treatment could inhibit the expressions of Non-p-β-catenin and CD133 in colonic organoids （P<0.01）. ConclusionQHJ could inhibit the inflammation-cancer development in CAC mice， the mechanism of which might be related to regulating the microenvironment and inhibiting the over-activation of Wnt signaling.