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??OBJECTIVE To develop an RP-HPLC method for simultaneous determination of rosmarinic acid, tilianin, luteolin-7-O-??-D-glucuronide, apigenin-7-O-??-D-glucuronide, and diosmetin-7-O-??-D-glucuronide in different parts of Dracocephalum moldevica L. METHODS The five constituents were measured on a Shim-pack ODS column(4.6 mm??250 mm, 5 ??m)with gradient elution of acetonitrile (A)-0.5% formic acid aqeous solution (B) (0-30 min, 17%A;30-60 min, 17%-28%A; 60-70 min, 28%A) at a flow rate of 1.0 mL??min^-1.The detection wavelength was set at 330 nm, and the column temperature was maitained at 35 ??. RESULTS The linear ranges of rosmarinic acid, tilianin, luteolin-7-O-??-D-glucuronide, apigenin-7-O-??-D-glucuronide, and diosmetin-7-O-??-D-glucuronide were 4.2-126 ??g??mL^-1 (r=0.999 2), 7.84-235.2 ??g??mL^-1 (r=0.999 3), 3.048-91.44 ??g??mL^-1(r=0.999 4), 1.472-44.16 ??g??mL^-1(r=0.999 4), and 2.816-84.48 ??g??mL^-1 (r=0.999 2), respectively.The average recoveries (RSD) of the five compounds were 98.97%(1.03%), 99.90%(0.92%), 99.89% (1.75%), 99.55% (0.98%), and 99.76%(1.19%) (n=6), respectively. CONCLUSION The developed method is accurate and precise, which can be used for the quality control of different parts of Dracocephalum moldevica L.The RESULTS of content determination indicate that the five compounds exist in all the parts of Dracocephalum moldevica L., but the mass fractions are obviously different.     

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??OBJECTIVE To establish a high-performance capillary electrophoresis (CE) method for determinating gallic acid in commercial Quanshen pieces of two different colors,amaranth and brownish red. METHODS CE-UV method was used in the following condition: BGE 20 mmol??L^-1 sodium borate solution, voltage 20 kV, detection wavelength 290 nm, sample injection 10 s at 5 kPa, capillary temperature room temperature. RESULTS A quantitative method was successfully developed to determine gallic acid in Quanshen pieces within 6 min. The contents of gallic acid in 7 batches of amaranth colored Quanshen pieces were 2.70% (Hubei province), 12.08% and 7.79% (Shandong province), 2.01%, 5.78%, 6.97%, and 4.22% (Hebei province), respectively. The contents of gallic acid in brownish red colored Quanshen pieces were 1.35% (Hubei province), 1.81% and 3.42% (Shandong province), 1.87%, 3.42%, 0.44%, and 1.52% (Hebei province), respectively. CONCLUSION The method is rapid, economical and simple and can provide a powerful quality control measure for Quanshen pieces. The contents of gallic acid in the amaranth colored Quanshen pieces are higher than those in the brownish red colored pieces.     

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??OBJECTIVE To discuss whether the difference in dissolution profile in vitro may cause different bioavailability in vivo and investigate the effects of the key quality parameters of leflunomide on bioavailability.METHODS Using SANOFI product as the reference preparation and domestic product as the test preparation, the disintegration solution of leflunomide tablets was analyze by Morphologi G3-ID automated measurement to get the paricile size and size distribution of the API; using pH 6.5 FaSSIF solution without adding ox-gall sulfonic acid sodium and lecithin as the dissolution medium, the dissolution and permeation profiles of the reference and test preparations and raw material were compared at 37 ?? with rotate speed of 150 r??min^-1. The influence of quality parameters on the process of API??s release and absorption was investigated, then the difference between the reference and test preparations were compared to preliminarily predict the bioavailability and bioequivalence.RESULTS The particle size Dv(50)of domestic leflunomide tablets was 79.80 ??m, while the particle size Dv(50)of the reference product was 17.60 ??m; the dissolution rate and penetration rate of the test preparation were about 70% of the the reference preparation, the t_max was basically identical,but the ??_max and AUC_0-t were lower than the reference preparation. The bioavailability of the test preparation was about 90% of the reference preparation.CONCLUSION Though the dissolution profile of domestic leflunomide tablets is not identical to the reference preparation, but the two products were predicted to be bioequivalent.     

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??OBJECTIVE To establish a method for determining the contents of saikosaponin a, b₁, b₂ and c in Radix Bupleuri formula granules by HPLC-MS/MS, and provide scientific basis for the quality standards. METHODS The samples were separated on a Kinetex C₁₈ column (2.1 mm??100 mm, 2.6 ??m) by gradient elution at the flow rate of 300 ??L??min^-1 using acetonitrile and 0.1% aqueous formic acid as the mobile phase. The column temperature was maitained at 40 ??. Multiple reaction monitoring scanning (MRM) was employed for the quantification with switching electrospray ion source polarity in negative mode. The ion spray voltage was set at -4 500 V and the turbo spray temperature was maintained at 550 ??. RESULTS There was significant correlation between the peak area and concentration of each compound within the test range (r??0.999 1). The average recoveries were from 97.44% to 100.56%. CONCLUSION Saikosaponin a, b₁, b₂ and c are determined by this method simultaneously. The method is available with good reproducibility and can control the quality of Radix Bupleuri formula granules effectively.     

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??OBJECTIVE To investigate antagonistic activities of three isomers of ??-conotoxin TxIB on rat and human ??6/??3??2??3 nicotinic acetylcholine receptors (nAChRs). METHODS Three disulfide bond isomers were synthesized using Fmoc chemistry, which were identified by ultra performance liquid chromatography (UPLC)and confirmed by MALDI-TOF mass spectrometry. Rat and human ??6/??3??2??3 nAChRs were expressed in oocytes of Xenopus laevis, which were used to test the antagonistic abilities of the 3 isomers. RESULTS The three isomers of ??-conotoxin TxIB were synthesized successfully.The retention time of each isomer of ??-conotoxin TxIB was different each other significantly. The observed molecular masses of three isomers were the same, which were consistent with their theoretical molecular mass.Their hydrophilicity orders were globular > ribbon> bead. Both rat and human ??6/??3??2??3 nAChRs were expressed in oocytes well. Inhibition of three isomers of ??-conotoxin TxIB on rat and human ??6/??3??2??3 nAChRs were evaluated respectively. Among the three isomers of TxIB, the activity of the globular isomer was the most potent one, which had almost same activity at rat and human ??6/??3??2??3 nAChRs with corresponding IC₅₀ of 28.2 and 32.0 nmol??L^-1respectively. However, the other two isomers, ribbon and bead isomers displayed little antagonistic effect on both rat and human ??6/??3??2??3 nAChRs only with an IC₅₀ of ??10 ??mol??L^-1. CONCLUSION The synthesized globular isomer of ??-conotoxin TxIB in this work has a high selectivity and potent antagonistic activity on rat and human ??6/??3??2??3 nAChRs, which would be helpful for its new drug development.     

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??OBJECTIVE To investigate the anti-depressant effect of schisandrin A in rats with depression caused by chronic unpredictable mild stress(CUMS)as well as the relevant mechanism. METHODS The depression-like rat model using CUMS was established. Rats were randomly divided into control, CUMS model, CUMS+fluoxetine (10 mg??kg^-1)and CUMS + schisandrin A (25, 50, 100 mg??kg^-1)groups. Drugs or vehicle were administrated after stress procedures for 21 d. Open-field test (OFT), sucrose preference tests (SPT)and forced swim test (FST)were used to evaluate the anti-depressant effects of schisandrin A. The reactive oxygen species (ROS)and prostaglandin E₂ (PGE₂) level as well as superoxide dismutase (SOD) and catalase (CAT)activities in hippocampus were determined by ELISA methods. IL-1??, TNF-??, and IL-10 expression were measured by real time qPCR and Western blot analysis. RESULTS Behavioral test indicated that crossing score and rearing score in OFT and sucrose preference index in SPT of model group were significantly lower than control group (P<0.01), while immobility time in FST was significantly increased (P<0.01). Compared with those in control group, the ROS and PGE₂ level increased significantly (P<0.01), SOD and CAT activities decreased significantly (P< 0.01),the mRNA and protein level of IL-1??, TNF-??, and IL-10 were increased significantly (P<0.05 or P<0.01)in rats of CUMS. CONCLUSION Schisandrin A and fluoxetine could ameliorate those changes induced by CUMS. Schisandrin A could improve the depression-like behaviors of rats induced by CUMS, of which the mechanism might involve the antioxidant and anti-inflammatory effects.     

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??OBJECTIVE To investigate the therapeutic effects of benzylbenzoate glucosides from Curculigo orchioides on retinoic acid induced osteoporosis in rats. METHODS 3-Month-old female Sprague-Dawley rats were intragastrically given the retinoic acid 70 mg??kg^-1??d^-1 for 2 weeks, and then treated with estrogen(E₂, 1 mg??kg^-1), total flavonoids from Epimedium folium(TF, 100 mg??kg^-1), benzylbenzoate glucosides from Curculigo orchioides (COP,6,18 and 54 mg??kg^-1) and Curculigo orchioides extracts(COE,3 000 mg??kg^-1)for 4 weeks, respectively. The bone mineral density of right femur were assayed by Dual Energy X-ray Absorptiometry. The levels of Ca, P, creatinine, ALP and TRAP in serum and urine were assayed with automatic biochemical analyzer. The levels of BGP and DPD in serum were measured with Elisa kit according to instruction. RESULTS Treatment SD rats with retinoic acid for 2 weeks significantly decreased bone mineral density, increased the ratio of Ca and Creatinine in urine, and serum TRAP activity. Treatment with E₂, TF and COP not only increased the bone mineral density, decreased the ratio of Ca and creatinine in urine and serum TRAP activity, but also regulated BGP level and increased ALP activity of serum in retinoic acid induced osteoporotic rats. CONCLUSION Benzylbenzoate glucosides from Curculigo orchioides can decrease bone loss by inhibiting bone resorption and improving bone formation.     

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??OBJECTIVE To establish the high performance liquid chromatography (HPLC) fingerprint of the caulis of Chimonanthus nitens and evaluate the product quality by chemometrics analysis method. METHODS The method was developed on an Amethyst C₁₈-H column(4.6 mm??250 mm, 5 ??m)by gradient elution with acetonitrile-water (containing 0.1% formic acid) as mobile phase at a flow rate of 0.8 mLmin^-1. The column temperature was maintained at 28 ??, and the detection wavelength was set at 254 nm. The main characteristic peaks was identified by comparing the retention time and UV absorption characteristics. Then 10 batches of the caulis of Chimonanthus nitens were evaluated by similarity assay, HCA, and PCA. RESULTS The HPLC fingerprint of the caulis of Chimonanthus nitens was established and three main peaks were identified. The similarity of 10 batches of the caulis of Chimonanthus nitens was about 0.978 0 to 0.991 9. CONCLUSION The established method can be used for the quality control of the caulis of Chimonanthus nitens.     

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??OBJECTIVE To establish an HPLC method for simultaneous determination of ginsenoside Rg₁, Re, Rb₁ and jujuboside A, B in Renshen Jianpi Pellets (RSJPW). METHODS The determination was conducted on an Eclipse XDB C₁₈ column (4.6 mm??250 mm, 5 ??m) with the mobile phase of acetonitrile (A)-water (B) gradiently eluted at the following flow rates:0-90 min, 1.0 mL??min^-1; 90-130 min, 1.0-0.5 mL??min^-1; 130-140 min, 0.5 mL??min^-1. And the column temperature was maintained at 35 ??; the detection wavelength was set at 203 nm. RESULTS The calibration curves of ginsenoside Rg₁, Re, Rb₁ and jujuboside A, B were in good linearity over 0.32-2.24 ??g (r=0.998 2), 0.16-1.12 ??g (r=0.995 3), 0.32-2.24 ??g (r=0.999 6), 0.16-1.12 ??g (r=0.991 5), 0.08-0.56 ??g (r=0.999 6). The corresponding average recovery rates were 97.18%, 97.62%, 98.79%, 98.48%, 94.51%; the standard deviations were 1.10%, 0.98%, 0.34%, 1.09%, 1.88%, respectively. CONCLUSION The established HPLC method is accurate, reliable and reproducible, which can be used as a reference for the quality control of RSJPW.     

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??A disintegrin-like and metalloprotease(ADAMTS) proteinases are a group of multidomains and secreted metalloproteinases containing the thrombospondin motifs. ADAMTS-7 is a member of ADAMTS family and plays a crucial role in the cardiovascular diseases. In physically, ADAMTS-7 was involved in the growth and development of the body. However, in pathological conditions, over expression of ADAMTS-7 gene accelerated the progression of cardiovascular diseases through by promoting the breakdown of cartilage oligomeric matrix protein (COMP) matrix. Meantime, the over expression of ADAMTS-7 can be effectively inhibited by TGF-??, miRNA29a/b, and then effectively retarded the development of cardiovascular disease. This article mainly reviews the gene structure, protein function and the mechanism of ADAMTS-7 involved in cardiovascular diseases and looks forward to the possibility of ADAMTS-7 as a new target for the treatment of cardiovascular diseases.     

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??OBJECTIVE To examine the effects of TPGS 1000 and Soluplus on the transport of ginsenoside CK in Caco-2 cell model. METHODS The effects of TPGS 1000 and Soluplus at different concentrations on ginsenoside CK were evaluated by using Caco-2 cell model. The concentration of ginsenoside CK in cell was examined by ultra high pressure liquid chromatography (UPLC) method. The apparent permeability coefficient (P_app) and the efflux ratio were calculated. RESULTS When the proportion of ginsenoside CK to TPGS 1000 or Soluplus was 1??1, 1??3, and 1??9, the absorption of ginsenoside CK significantly increased. The efflux and efflux ratio both decreased significantly(P<0.05). TPGS 1000 had more significant promotion effect on the transport of ginsenoside CK than the same dose of soluplus at the same ratio (P<0.05). CONCLUSION In Caco-2 cell model, both TPGS 1000 and Soluplus can significantly promote the absorption of ginsenoside CK.
     

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??OBJECTIVE To study the active ingredients of Flos Carthami by investigating the relationship between HPLC spectrum and antioxidant activity. METHODS The specific chromatogram of Flos Carthami processed by means of classical homothermal acceleration was assayed by HPLC. DPPH, ABTS and FRAP assays were established to determine the antioxidant activity of Flos Carthami. The spectrum-effect correlation was studied by partial least squares (PLS) method. RESULTS S₄, S₈, S₁₁, S₁₂, S₁₃, S₁₄, S₁₆ and S₁₇ were characteristic compounds in 27 matching characteristic chromatograms which had positive relationship with the ability of scavenging DPPH free radical. S₄, S₈, S₁₁, S₁₂, S₁₄, S₁₆, S₁₇ and S₁₈ were significantly positively related to the ability of scavenging ABTS free radical. S₄, S₈, S₁₁, S₁₂, S₁₃, S₁₄, S₁₆, S₁₇, S₁₈ and S₂₆ were significantly positively related to the ability of restoring Fe³⁺. Among the chromatographic peaks, S₄, S₈, S₁₁, S₁₂, S₁₄, S₁₆ and S₁₇ were positively related to the abilities of scavenging DPPH free radical and ABTS free radical and restoring Fe³⁺. S₁₄ was determined as hydroxysafflor yellow A. CONCLUSION The antioxidant material of Flos Carthami is based on the synergy of a variety of compounds. An as comprehensive as possible analysis of the compounds related to the pharmacological effect can reflect the quality of Flos Carthami. The study of spectrum-efficacy relationship is an effective way to determine the compounds related with pharmacological effect and control the quality of traditional Chinese medicinal materials.     

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??OBJECTIVE To interrogate differential sensitivity of ??-conotoxin TxID on stoichiometry of ??3??4 nicotinic acetylcholine receptors(nAChRs). METHODS Oocytes of Xenopus laevis were used to express rat ??3??4 nAChRs with different stoichiometries by altering ??3:??4 RNA injection ratios of 1:1, 1:10 or 10:1. Sensitivity of ??-conotoxin TxID on these different stoichiometry receptors were evaluated and compared. RESULTS The three stoichiometry receptors of ??3??4 nAChRs were expressed in oocytes successfully. ??-Conotoxin TxID showed differential sensitivity on ??3??4 nAChR stoichiometries. Inhibition of 1:10 injection ratio by TxID was similar with regular 1:1 ??3??4 nAChRs within 2-fold difference. While potency of 10:1 injection ratio by TxID decreased 5-fold significantly comparing with 1:1 ??3??4 nAChRs. CONCLUSION ??-Conotoxin TxID exhibits distinct sensitivity on different stoichiometry of ??3??4 nAChRs, which could reflecting different stoichiometries of ?? and ?? subunits. The RESULTS would be helpful for elucidation of structure and physiology function of ??3??4 nAChRs.     

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??OBJECTIVE To perform a multidimensional study on the critical quality attributes of a therapeutic anti-HER2 humanized monoclonal antibody targeting HER2-positive breast cancer. METHODS The critical quality attributes such as purity, impurities, structure, and function were evaluated using multidimensional analytical techniques including HPLC, CE-SDS, dynamic light scattering,circular dichroism,differential scanning calorimeter, LC-MS/MS, in vitro bioactivity assay and SPR binding kinetics. RESULTS The monomer content of the antibody was more than 98% while the polymer impurities were less than 2%. The sum of heavy chain and light chain peak area was over 97% while the non-glycosylated heavy chain impurities area was less than 1% on the CE-SDS electrophoretogram. The antibody drug demonstrated comparable CD spectrum, predicted secondary structure and unfolding temperatures to the reference product. It had highly similar N-glycan profile involving glycosylation site and N-glycan types to the reference product. The analysis showed no significant difference in the functional CQAs like BT474 proliferation inhibiting bioactivity, ADCC efficacy,the binding affinity to HER2 and Fc receptors between the evaluated drug product and the reference. CONCLUSION The characteristics of the monoclonal antibody drug such as high purity and few impurities indicate homogeneity and low-risk of safety issues. High similarity to the reference has been verified in multiple aspects like higher structure, glycosylation and function. The positive quality evaluation result is a prerequisite for clinical research. Moreover,to some extent, there can be potential correlation between CQAs and clinical safety and efficacy.     

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??OBJECTIVE To develop a simultaneous quantitative analysis method of four triterpenoids in Alismatis Rhizoma by multi-components by single maker (QAMS). METHODS Four main triterpenoids (alisol A, alisol A 24-acetate, alisol B and alisol B 23-acetate) were selected as analytes to evaluate the quality of Alismatis Rhizoma. Alisol B 23-acetate was used as the internal reference standard and three relative correction factors (RCFs) to alisol B 23-acetate were calculated. These RCFs were obtained by high performance liquid chromatography coupled with diode array detector under various chromatographic conditions. Then 31 batches of Alismatis Rhizoma samples collected from different areas were determined by both external standard method (ESM) and QAMS. RESULTS The RCFs were obtained with good reproducibility (RSD ?? 2.25%). No obvious differences were observed between the analysis results of QAMS method and ESM method (RSD ?? 3.52%). CONCLUSION The established QAMS method is reliable and feasible for simultaneous analysis of four triterpenoids and could be used for quality control of Alismatis Rhizoma.     

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??OBJECTIVE To determine simultaneously the contents of xanthotoxin,bergaptol, and bergapten in cultivated Changium smyrnioides Wollf and its in vitro cultures by HPLC.METHODS Agilent-C₁₈ column (4.6 mm??250 mm,5 ??m) was used for chromatographic separation and PAD was applied as detector. The flow rate was 1.0 mL??min^-1with a mobile phase of methanol-water in gradient elution mode. The detection wavelength was set at 314 nm while the injection volume was 10 ??L.RESULTS The linear regression equations of xanthotoxin, bergaptol, and bergapten were Y=17 057??-87.689 (r=1.000 0), Y=23 828??-380.44 (r=0.999 9) and Y=37 123??-441.16(r=0.999 9), respectively. The contents of xanthotoxin and bergapten in cultivated and regenerated specimens were obviously higher than those in calli and suspension cells, while bergaptol was only detected in the latter two samples. A larger amount of furanocoumarins accumulated in the cells from leaves and petioles. CONCLUSION The established method is simple and effective with high sensitivity and good repeatability. It can be adopted in studies on the utilization of Changium smyrnioides, especially the regulation and induction of furanocoumarins.     

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??OBJECTIVE To establish an RP-HPLC method for determination of the contents of eplerenone and its related substances. METHODS A RP-HPLC method was developed by using a C₁₈ column (Agilent Poroshell,4.6 mm??100 mm, 2.7 ??m), the mobile phase consisted of H₂O-CH₃CN-MeOH (64??18??18), the flow rate was 1.0 mL??min^-1, and the detection wavelength was set at 240 nm. The diluent was H₂O-CH₃CN-MeOH (50??25??25). RESULTS Eplerenone was well separated from the impurities. For the determination of related substances, the LODs of eplerenone, its intermediate (?? and ??) and related substances (A-E) were 0.03, 0.13, 0.25, 0.05, 0.05, 0.26, 0.07 and 0.12 ng, and the LOQs were 0.12, 0.42, 0.84, 0.18, 0.18, 0.86, 0.22 and 0.41 ng, respectively. The calibration curves of eplerenone and related substances (A-E) had good linear relationship within 0.13-1.53, 0.25-3.03, 0.25-2.99, 0.13-1.61, 0.14-1.63, and 0.13-1.54 ??g??mL^-1(r=0.999 9, n=7), and the correction factors of the related substances (A-E) were all in the range of 0.90-1.10. The average recoveries of the related substances (A-E) were all within 95.0%-105.0% (n=9). For the content determination of eplerenone, the validation test showed good linearity over the range of 12.60-75.63 ??g??mL^-1 (r=0.999 6, n=6) with good repeatability (RSD=0.19%, n=6). CONCLUSION The developed method proves to be simple, accurate, specific and reliable. It can be applied to the determination of eplerenone and its related substances.     

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??Hepatocellular carcinoma(HCC) is a common malignancy,of which the 5-year survival rate is 39% merely. HCC current treatments include surgery, interventional chemotherapy, radiofrequency ablation, and targeting drug delivery. As a typical hypervascular cancer, HCC can be inhibited via tumor angiogenesis inhibitors(TAI). However, TAI can not completely block the nutrient supply of the tumor tissue during treatment, since there exists a special way of blood supply named vascular mimicry(VM) in HCC. Through these pipes similar to the blood vessels HCC can communicate with the host blood vessels and thus acquire blood supply for the growth, invasion and metastasis. A growing number of studies have found that the presence of angiogenesis mimicry structure is one of the key factors limiting TAI in the treatment of liver cancer. This article gave an outline of VM and summarized the formation mechanism of VM and the research status about the hepatocarcinoma therapy.     

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??OBJECTIVE To investigate the impact factors of the semi-quantitative Western blot (WB), offering a guidance on research of pharmacological effects of drugs at the level of proteins. METHODS Total proteins were extracted from the Huh7.5 cells infected with hepatitis C virus and quantified with BCA kit, HCV Core protein was chose as target protein, and protein Tubulin or Gapdh, which was encoded by housekeeping gene, as the internal control. By controlling the experimental factors, such as loading sample amount, concentration and the incubation time of first antibodies, and dilutions of chemiluminescent fluid as well, we analyzed those factors how to impact the semi-quantitative results. RESULTS The semi-quantitative results showed that there is a linear relationship between relative intensity of target protein and the amount of total protein at must protein concentration range, beyond which, the rangeability of relative intensity of target protein reduced, even no changes. However, sample volume loaded, or protein selected as internal reference has little influence on the result of semi-quantitative WB. In the context of obtaining high-quality band, concentration and incubation time of antibodies or dilution of chemiluminescent fluid also has no influence on it. Yet, the semi-quantitative WB has certain defects and our results show that the permissible error of semi-quantitative result should be controlled within 15%. CONCLUSION The key impact factor on the result of semi-quantitative WB is the target protein amount of loading. On the premise of obtaining clear and high-quality bands, appropriately selecting loading volume of samples, concentration and incubation time of first antibodies, and dilutions of chemiluminescent fluid is conducive to accomplish experiments without affecting the final results of semi-quantitative WB.