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ObjectiveTo study the inhibitory effect of polyphyllin Ⅰ （PPI） on the growth of colorectal cancer cells and its molecular mechanism. MethodRKO cells were cultured and divided into a blank group and PPI treatment groups with concentrations of 0.6， 0.8， 1.0 μmol·L-1， respectively. HRT18 cells were cultured and divided into a blank group and PPI treatment groups with concentrations of 1.2， 1.4， 1.6 μmol·L-1， respectively. The effects of PPI on the proliferation and morphology of colorectal cancer were detected by cell proliferation toxicity assay， trypan blue exclusion assay， plate clone formation assay， and confocal high-intension cell imaging analysis system. Flow cytometry was used to detect the apoptosis rate of colorectal cancer cells. The pQCXIP-GFP-LC3 plasmid transfection assay was used to detect the formation of autophagosomes in colorectal cancer cells after PPI treatment. Western blot was used to detect the expression of apoptosis-related proteins Caspase-3， Caspase-8， and poly ADP ribose polymerase （PARP）， the expression of autophagy related protein LC3Ⅱ， and the expression and phosphorylation of Hippo signaling pathway proteins LATS1 and YAP. In the plvx-Flag-YAP plasmid transfection assay， YAP was overexpressed and treated with PPI， and the proliferation of colorectal cancer cells was detected by cytotoxicity assay. The expression of LC3Ⅱ and PARP in colorectal cancer cells was detected by Western blot. SwissADME predicted pharmacokinetic parameters of PPI. ResultAs compared with the blank group， the survival rate and clone formation ability of colorectal cancer cells in the PPI group were significantly decreased （P<0.01）， the cell area of colorectal cancer cells in the PPI group was significantly decreased， and the roundness of colorectal cancer cells was significantly increased （P<0.01）. As compared with the blank group， the apoptosis rate of colorectal cancer cells in PPI treatment groupw was significantly increased （P<0.01）， the expression of apoptotic proteins Caspase-3 and Caspase-8 protein precursor in PPI treatment groups was decreased， and the cleavage of PARP was increased （P<0.01）. As compared with the blank group， the expression level of autophagy-related protein LC3Ⅱ in colorectal cancer cells in PPI treatment groups was significantly increased， and the formation of autophagosomes was promoted （P<0.01）. As compared with the blank group， the expression of YAP protein in colorectal cancer cells in PPI treatment groups was significantly decreased， and the expressions of phosphorylated LATS1 and YAP were significantly increased （P<0.01）. As compared with the blank group， overexpression of YAP could significantly antagonize the effect of PPI on apoptosis， autophagy activation， and proliferation inhibition of colorectal cancer cells. SwissADME simulation results showed that PPI had good drug like activity. ConclusionPPI can induce apoptosis and autophagy of colorectal cancer cells through targeted activation of Hippo signaling pathway， thereby inhibiting their proliferation.
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Polyphyllin Ⅰ(PPⅠ)and polyphyllin Ⅱ(PⅡ)are the main active substances in the Paris polyphylla.However,liver toxicity of these compounds has impeded their clinical application and the potential hepatotoxicity mechanisms remain to be elucidated.In this work,we found that PPⅠ and PⅡ exposure could induce significant hepatotoxicity in human liver cell line L-02 and zebrafish in a dose-dependent manner.The results of the proteomic analysis in L-02 cells and transcriptome in zebrafish indicated that the hepa-totoxicity of PPⅡ and PⅡwas associated with the cholesterol biosynthetic pathway disorders,which were alleviated by the cholesterol biosynthesis inhibitor lovastatin.Additionally,3-hydroxy-3-methy-lglutaryl CoA reductase(HMGCR)and squalene epoxidase(SQLE),the two rate-limiting enzymes in the choles-terol synthesis,selected as the potential targets,were confirmed by the molecular docking,the over-expression,and knockdown of HMGCR or SQLE with siRNA.Finally,the pull-down and surface plasmon resonance technology revealed that PPⅠ could directly bind with SQLE but not with HMGCR.Collectively,these data demonstrated that PPⅠ-induced hepatotoxicity resulted from the direct binding with SQLE protein and impaired the sterol-regulatory element binding protein 2/HMGCR/SQLE/lanosterol synthase pathways,thus disturbing the cholesterol biosynthesis pathway.The findings of this research can contribute to a better understanding of the key role of SQLE as a potential target in drug-induced hepatotoxicity and provide a therapeutic strategy for the prevention of drug toxic effects with similar structures in the future.
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Paridis Rhizoma， a traditional valuable Chinese herbal medicine， has the functions of clearing heat and removing toxin， relieving edema and pain， cooling liver and calming convulsion， which can be used to treat various diseases such as mumps， abscess， burn， bleeding， and tumor. It has been used in folk medicine for a long time and is the main raw material of various Chinese patent medicines such as Gongxuening Capsules and Yunnan Baiyao. Polyphyllin Ⅰ， an isospirostanol saponin and one of the main active components in Paridis Rhizoma， is distributed in the rhizome， pericarp， and leaves of Paris polyphylla. With high polarity， polyphyllin Ⅰ is mainly extracted by n-butanol extraction and macroporous adsorption resin chromatography， separated by silica gel column chromatography and preparative high performance liquid chromatography， and purified with the combination of methods. With anti-tumor， anti-inflammatory， antibacterial， and anti-virus effects， it is generally employed to treat liver cancer， lung cancer， gastric cancer and other cancers as well as arthritis， influenza， sore toxin， and bacterial infection. However， polyphyllin Ⅰ may cause stomach irritation， hemolysis， liver damage， kidney damage， heart damage， and other adverse reactions. Pharmacokinetic studies show that it has problems such as low bioavailability and poor intestinal absorption and permeability， which affect the clinical application of polyphyllin Ⅰ. This paper summarizes the research on the plant sources， extraction and separation methods， pharmacological effects， adverse reactions， and pharmacokinetics of polyphyllin Ⅰ in recent years， which is expected to provide a reference for the rational clinical application and other in-depth research work of polyphyllin Ⅰ.
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This study aims to investigate the molecular mechanism of polyphyllin Ⅰ(PPⅠ) inhibiting proliferation of human breast cancer cells. Human breast cancer BT474 and MDA-MB-436 cells were treated with different concentrations of PPⅠ, and then the effect of PPⅠ on cell proliferation was detected by MTT assay, trypan blue dye exclusion assay, real-time cell analysis, and clone forming assay, respectively. The apoptosis was detected by Annexin V-FITC/PI staining and then analyzed by flow cytometry. The change of mitochondrial membrane potential was detected by flow cytometry after fluorescent probe JC-1 staining. Western blot was used to detect protein expression and phosphorylation. Molecular docking was performed to detect the binding between PPⅠ and EGFR. The affinity between PPⅠ and EGFR was determined by drug affinity responsive target stability assay. The results indicated that PPⅠ inhibited the proliferation and colony formation of BT474 and MDA-MB-436 cells in a time-and concentration-dependent manner. The PPⅠ treatment group showed significantly increased apoptosis rate and significantly decreased mitochondrial membrane potential. PPⅠ down-regulated the expression of pro-caspase-3 protein, promoted the cleavage of PARP, and significantly reduced the phosphorylation levels of EGFR, Akt, and ERK. Molecular docking showed that PPⅠ bound to the extracellular domain of EGFR and formed hydrogen bond with Gln366 residue. Drug affinity responsive target stability assay confirmed that PPⅠ significantly prevented pronase from hydrolyzing EGFR, indicating that PPⅠ and EGFR have a direct binding effect. In conclusion, PPⅠ inhibited the proliferation and induced apoptosis of breast cancer cells by targeting EGFR to block its downstream signaling pathway. This study lays a foundation for the further development of PPⅠ-targeted drugs against breast cancer.
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Female , Humans , Apoptosis , Breast Neoplasms/genetics , Cell Line, Tumor , Cell Proliferation , Diosgenin/analogs & derivatives , ErbB Receptors , Molecular Docking SimulationABSTRACT
Objective:To observe the effect of polyphyllin Ⅰ on the expressions of forkhead box Q1（FOXQ1）and epithelial-mesenchymal transition (EMT)-related factors, in order to explore the possible mechanism of polyphyllin Ⅰ in inhibiting the metastasis of colon cancer. Method:After the treatment with 1.25，2.50 μmol·L-1 polyphyllin Ⅰ on HCT116 cells, Western blot and Real-time PCR were used to detect the expressions of FOXQ1，E-cadherin，Vimentin protein and mRNA. Result:Compared with the blank group, relative expressions of FOXQ1 protein and mRNA in low-concentration polyphyllin Ⅰ group were decreased, while relative expression of E-cadherin mRNA was increased, the differences were not statistically significant, and relative expressions of Vimentin protein and mRNA in low-concentration polyphyllin Ⅰ group were decreased (P<0.05)， and relative expression of E-cadherin protein in low-concentration polyphyllin Ⅰ group was increased (P<0.01). Compared with the blank group, relative expressions of FOXQ1, Vimentin protein and mRNA in high-concentration polyphyllin Ⅰ group were decreased，while relative expressions of E-cadherin protein and mRNA were increased (P<0.05, P<0.01). Compared with low-concentration polyphyllin Ⅰ group, relative expressions of Vimentin protein and mRNA in high-concentration polyphyllin Ⅰ group were decreased, but the difference was not statistically significant, relative expressions of E-cadherin protein and mRNA in high-concentration polyphyllin Ⅰ group were increased, whereas relative expressions of FOXQ1 protein and mRNA were decreased (P<0.05, P<0.01). Conclusion:Mechanism of polyphyllin Ⅰ inhibiting the metastasis of colon cancer may be related to the decrease of FOXQ1 and Vimentin expressions, and the up-regulation of E-cadherin.
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OBJECTIVE: To study the fingerprints of rhizomes of Paris polyphylla Smith var. polyphylla and Paris polyphylla Smith var. yunnanensis (Franch.) Hand. -Mazz from different origins in Dali and the differences of seven main steroidal saponins. METHODS: The fingerprints of rhizomes of Paris polyphylla var. polyphylla and Paris polyphylla var. yunnanensis from different origins in Dali were established by HPLC. The similarity of fingerprints and seven main steroidal saponins were compared and analyzed. RESULTS: There were 14 common peaks in the fingerprints of rhizomes of Paris polyphylla var. polyphylla from different origins in Dali, and the similarity of the fingerprints of the samples from different habitats except Jinhua Weishan and Longjie Weishan was greater than 0.9. There were 13 common peaks in the fingerprints of rhizomes of Paris polyphylla var. yunnanensis from different origins in Dali, and the similarity of the samples from different origins was greater than 0.92. There were 11 common peaks in the fingerprints of rhizomes of Paris polyphylla var. polyphylla and Paris polyphylla var. yunnanensis from different origins in Dali, and the similarity of the fingerprints was very low, between 0.057 and 0.225. Polyphyllin Ⅰ, polyphyllin Ⅱ, polyphyllin Ⅵ and polyphyllin Ⅶ are detected in the rhizomes of Paris polyphylla var. polyphylla and Paris polyphylla var. yunnanensis from different origins in Dali. The average peak areas of polyphyllin Ⅰ, polyphyllin Ⅱ, polyphyllin Ⅵ and polyphyllin Ⅶ in Paris polyphylla var. yunnanensis from different origins in Dali were higher than those of Paris polyphylla var. polyphylla. Polyphyllin H was detected in the rhizomes of Paris polyphylla var. yunnanensis from Leqiu Nanjian, Jinhua Weishan, Fengyu Eryuan, Dengchuan Eryuan, Hongyan Midu, Xiyi Heqing and the rhizomes of Paris polyphylla var. polyphylla from Wanqiao Dali, Fengyi Dali, Xiyi Heqing, Hongyan Midu and Leqiu Nanjian. Polyphyllin Ⅲ was detected in the rhizomes of Paris polyphylla var. polyphylla from the origins except Longjie Weishan. Polyphyllin Ⅴ was also detected in the rhizomes of Paris polyphylla var. polyphylla in Fengyu Eryuan and Xiyi Heqing. CONCLUSION: Polyphyllin Ⅰ, polyphyllin Ⅱ, polyphyllin Ⅵ and polyphyllin Ⅶ are detected in the rhizomes of Paris polyphylla var. polyphylla and Paris polyphylla var. yunnanensis from different origins in Dali. The similarity of the HPLC fingerprints of the rhizomes of Paris polyphylla var. polyphylla and Paris polyphylla var. yunnanensis is very low, and there are great differences in seven main steroidal saponins. The contents of polyphyllin Ⅰ, polyphyllin Ⅱ, polyphyllin Ⅵ and polyphyllin Ⅴ in Paris polyphylla var. yunnanensis are higher than those in Paris var. polyphylla.
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@#AIM: To observe the effects of high glucose-induced environment on Visfatin expression in human retinal pigment epithelial cells and to study the effects of Polyphyllin I on Visfatin expression in high glucose environment. <p>METHODS: Human retinal pigment epithelial cells were cultured in three groups, normal control group, high glucose group and intervention group of high glucose aggravated PolyphyllinⅠ, testing after 12h of intervention culture. Normal control group:5.5mmol/L glucose concentration routine culture; high glucose group:25mmol/L high glucose was added to the medium to establish the model; high glucose aggravated PolyphyllinⅠdrug intervention group: high glucose 25mmol/L, 3μg/L PolyphyllinⅠdrug was added to the medium. Immunofluorescence staining assays to observe expression of the Visfatin and VEGF in human retinal pigment epithelial cells; real-time PCR assays for relative expression of Visfatin and VEGF mRNA in epithelial cells; and western-blot assays for Visfatin and VEGF proteins in epithelial cells. <p>RESULTS: Immunofluorescence detection revealed that Visfatin and VEGF were weakly positive in normal retinal pigment epithelial cells. Visfatin and VEGF were strongly positive in high glucose group. Visfatin and VEGF fluorescence in the drug intervention group was significantly weakened in the higher sugar group. RT-PCR showed that the expression level visfatin mRNA high sugar group was significantly higher than that of normal group and intervention group(<i>t</i>=4.24, 3.89, <i>P</i><0.05). VEGF mRNA expression was significantly higher in high glucose group than in normal group and intervention group(<i>t</i>=3.53, 2.57, <i>P</i><0.05). Western-blot results showed that the protein expression of visfatin and VEGF in high sugar group was significantly higher than that in control group and intervention group(<i>t</i>=3.62, <i>P</i>=0.01; <i>t</i>=3.79, <i>P</i><0.01). <p>CONCLUSION: The high glucose environment can stimulate the increased expression of Visfatin in retinal pigment epithelial cells, Polyphyllin I can inhibit the expression of Visfatin in retinal pigment epithelial cells in high glucose environment, which may provide a new idea for the treatment of diabetic retinopathy.
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Objective To investigate the protective effect of PolyphyllinⅠ(PPI) on myocardial ischemia/reperfusion (I/R) injury in rats and its mechanism. Methods The 6-month-old Sprague-Dawley (SD) rats were divided into sham operation group (Sham group), I/R model group, and low, medium, high dose PPI groups according to the random number table method, with 10 in each group. The rat myocardial I/R model was prepared by ligating the left anterior descending branch of the coronary artery by 30 minutes and reperfusion by 120 minutes. Sham group was exposure to open chest without ligation. Low, medium, high dose PPI groups were injected with PPI 75, 150, 300 mg·kg-1·d-1 in front of the film for 4 weeks; dimethyl sulfoxide (DMSO) was gastric infused in the I/R model group. After the end of reperfusion, the myocardial infarction area (IA) was determined by triphenyltetrazole (TTC) and Evans blue (EB) staining;the apoptosis of myocardial cells was detected by TdT-mediated dUTP nick end labeling stain (TUNEL); the expressions of apoptosis related protein (Bax, Bcl-2), and cytoplasmic and nucleus expressions of P65 in nuclear factor-κB (NF-κB) signal pathway were detected by Western Blot. Results Compared with the Sham group, the myocardial IA was significantly increased in the I/R model group, the apoptosis rate of myocardial cells was significantly increased, the expression of Bcl-2 was significantly decreased, and the expression of Bax was significantly increased, and the intranuclear transfer of P65 was significantly increased. Compared with the I/R model group, low, medium and high dose PPI pretreatment could significantly reduce the myocardial IA [(21.6±0.9)%, (14.3±1.6)%, (15.0±0.8)% vs. (29.6±1.4)%], the apoptosis rate of myocardial cells was significantly decreased [(38.6±1.9)%, (24.3±2.6)%, (26.3±2.8)% vs. (56.8±2.4)%], the protein expression of Bcl-2 was significantly increased, while the protein expression of Bax was significantly decreased (Bcl-2/GAPDH: 0.24±0.07, 0.36±0.02, 0.34±0.09 vs. 0.13±0.04; Bax/GAPDH: 0.39±0.10, 0.21±0.08, 0.23±0.06 vs. 0.53±0.12); and P65 nuclear transfer was significantly decreased after middle and high dose PPI pretreatment [nuclear P65/Histone 3: 0.49±0.09, 0.51±0.06 vs. 0.83±0.11; cytoplasmic P65/GAPDH: 0.31±0.03, 0.30±0.05 vs. 0.22±0.07], with statistically significant differences (all P < 0.05). However, there was no significant difference in each index between the medium and high dose PPI groups (all P > 0.05). Conclusion PPI alleviates myocardial I/R injury in rats via NF-κB signal pathway, and the PPI effect of 150 mg·kg-1·d-1 is most especially significant.
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<p><b>Objective</b>To explore the inhibitory effect of polyphyllin Ⅰ (PPⅠ) on the proliferation of castration-resistant prostate cancer PC3 cells and its molecular mechanism.</p><p><b>METHODS</b>We cultured human prostate cancer PC3 cells in vitro and treated them with PPⅠ at the concentrations of 0 (blank group), 0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 μmol/L for 24, 48, and 72 hours, respectively. Then we detected the proliferation of the cells by MTT assay, measured their apoptosis by flow cytometry, and determined the expressions of p-ERK1/2, ERK1/2, NF-κB/p65 and DNMT1 proteins as well as the level of NF-κB/p65 in the cells additionally treated with the ERK1/2 inhibitor SP600125 by Western blot.</p><p><b>RESULTS</b>Compared with the blank control group, the PPⅠ-treated PC3 cells showed a concentration- and time-dependent reduction of the survival rate (1.00 ± 0.00 vs 0.85 ± 0.05, P < 0.01) at 0.4 μmol/L after 48 hours of intervention, concentration-dependent early apoptosis at 0.8 μmol/L (4.83 ± 0.95 vs 13.83 ± 2.97, P < 0.01), time-dependent increase of the expressions of p-ERK1/2 (1.00 ± 0.00 vs 1.73 ± 0.17, P < 0.01) and ERK1/2 (1.00 ± 0.00 vs 1.36 ± 0.12, P < 0.01) at 2 hours, and concentration-dependent decrease of the expressions of NF-κB/p65 and DNMT1 at 1.2 μmol/L (1.00 ± 0.00 vs 0.78 ± 0.10 and 0.63 ± 0.06, P < 0.01) and 1.6 μmol/L (1.00 ± 0.00 vs 0.67 ± 0.11 and 0.52 ± 0.09, P<0.01). Inhibition of ERK1/2 phosphorylation with PD98059 markedly reversed PPⅠ-induced decrease of the NF-κB/p65 expression as compared with that in the PPⅠ group (0.86 ± 0.18 vs 0.43 ± 0.09, P < 0.05).</p><p><b>CONCLUSIONS</b>PPⅠ induces the early apoptosis and suppresses the proliferation of PC3 cells, probably by activating the ERK1/2 pathway and inhibiting the expressions of the NF-κB/p65 and DNMT1 proteins.</p>
Subject(s)
Humans , Male , Apoptosis , Cell Proliferation , DNA (Cytosine-5-)-Methyltransferase 1 , Metabolism , Diosgenin , Pharmacology , Flavonoids , Metabolism , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 1 , Metabolism , Mitogen-Activated Protein Kinase 3 , Metabolism , NF-kappa B , Metabolism , PC-3 Cells , Phosphorylation , Prostatic Neoplasms, Castration-Resistant , Drug Therapy , Metabolism , Pathology , Signal Transduction , Transcription Factor RelA , MetabolismABSTRACT
Objective To investigate effects of polyphylin Ⅰ on the proliferation and apoptosis of human melanoma cell line A375,and to explore their mechanisms.Methods Normal human melanocytes isolated from healthy human foreskin were divided into 6 groups to be treated with 0,1.5,3.0,6.0,9.0,12.0 mg/L polyphyllin Ⅰ respectively.A375 melanoma cells were divided into 4 groups,i.e.,control group,1.5-,3.0-,6.0-mg/L polyphyllin Ⅰ groups,to be treated with 0,1.5,3.0,6.0 mg/L polyphyllin Ⅰ,respectively.Cell counting kit-8 (CCK8) assay was performed to evaluate the effect of polyphyllin Ⅰ on the proliferation of normal human melanocytes and A375 cells.Hoechst 33258 fluorescent staining was conducted to observe the morphology of apoptotic cells,flow cytometry to estimate cell cycle phase distribution and apoptosis rate,dichloro-dihydro-fluorescein diacetate (DCFH-DA) fluorescent probe assay to detect the level of reactive oxygen species (ROS),rhodamine-123 staining to evaluate changes of mitochondrial membrane potential,spectrophotography to detect the level of ATP in A375 cells,as well as levels of lactic acid and glucose in the culture supernatant of A375 cells,and Western blot analysis to determine the protein expression of Bcl-2,Bcl-2-related X protein (Bax),cleaved-caspase-3,cyclin D1 and pyruvate kinase isozyme type M2 (PKM2).Statistical analysis was carried out by using one-way analysis of variance (ANOVA) for comparisons among groups and Student-Newman-Keuls-q (SNK-q) test for multiple comparisons.Results CCK8 assay showed that the treatment with polyphyllin Ⅰ at concentrations of 1.5,3.0,6.0 mg/L for 48 hours had no effects on the proliferation of normal human melanocytes,but significantly inhibited the proliferation of A375 cells.The survival rate of A375 cells was significantly lower in the 1.5-,3.0-,6.0-mg/L polyphyllin Ⅰ groups than in the control group (P ＜ 0.01).After the treatment with polyphyllin Ⅰ,distinct apoptotic morphology of A375 cells was observed under fluorescence microscope.Additionally,along with the increase of polyphyllin Ⅰ concentrations (0,1.5,3.0,6.0 mg/L),there were gradual increasing trends in the apoptosis rate of A375 cells (4.25％ ± 1.27％,10.03％ ± 1.49％,36.62％ ± 1.97％,44.11％ ± 2.47％ respectively,F =665.7,P ＜ 0.01),the percentage of A375 cells at G0/G1 phase (54.13％ ± 2.57％,67.35％ ± 3.79％,74.39％ ± 3.29％,82.29％ ± 3.99％ respectively,F =71.81,P ＜ 0.01),the level of ROS in A375 cells (P ＜ 0.01),the level of glucose in the culture supernatant (P ＜ 0.01),and the protein expression of Bax and cleaved-caspase-3 (both P ＜ 0.01),while gradual decreasing trends were found in the levels of mitochondrial membrane potential and ATP in A375 cells (both P ＜ 0.01),the level of lactic acid in the culture supernatant (P ＜ 0.01),and the protein expression of Cyclin D1,Bcl-2 and PKM2 (all P ＜ 0.01).Conclusion Polyphyllin Ⅰ can effectively induce A375 cell apoptosis by promoting the production of ROS in A375 cells and decreasing the mitochondrial membrane potential,and arrest A375 cells at G0/G1 phase by inhibiting the expression of PKM2 and Cyclin D1.
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Objective To establish a method for determining the contents of polyphyllin Ⅰ and polyphyllin Ⅱ in Kangkeling Mixture. Methods The contents of polyphyllin Ⅰ and Ⅱ were determined by HPLC gradient elution. Poroshell 120 Ec-C18 column (4.6 mm × 150 mm, 4 μm) was used; Acetonitrile-water (A:B) was set as the mobile phase; the flow rate was 1.0 mL/min; the detection wavelength was 210 nm; column temperature was 30 ℃. Results Polyphyllin Ⅰ showed good linear relationship in the range of 1.009–10.09 μg (r=0.999 6), and the average recovery was 97.31% (RSD=2.05%, n=6). Polyphyllin Ⅱ showed good linear relationship in the range of 0.640 5–6.405 μg (r=0.999 8), and the average recovery was 96.41% (RSD=1.67%, n=6). Conclusion The method is simple, with good repeatability and accurate results, which can be used to determine the contents of polyphyllin Ⅰ and Ⅱ in Kangkeling Mixture.
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Objective To establish a method for determining the contents of polyphyllin Ⅰ and polyphyllin Ⅱ in Kangkeling Mixture. Methods The contents of polyphyllin Ⅰ and Ⅱ were determined by HPLC gradient elution. Poroshell 120 Ec-C18 column (4.6 mm × 150 mm, 4 μm) was used; Acetonitrile-water (A:B) was set as the mobile phase; the flow rate was 1.0 mL/min; the detection wavelength was 210 nm; column temperature was 30 ℃. Results Polyphyllin Ⅰ showed good linear relationship in the range of 1.009–10.09 μg (r=0.999 6), and the average recovery was 97.31% (RSD=2.05%, n=6). Polyphyllin Ⅱ showed good linear relationship in the range of 0.640 5–6.405 μg (r=0.999 8), and the average recovery was 96.41% (RSD=1.67%, n=6). Conclusion The method is simple, with good repeatability and accurate results, which can be used to determine the contents of polyphyllin Ⅰ and Ⅱ in Kangkeling Mixture.