| Malignant tumor is a serious disease that threatens human health.However,most of the current anti-tumor drugs have some unavoidable shortcomings,such as weak selectivity and severe side effects.Therefore,it is an urgent assignment to find natural anti-tumor agents with high efficiency and low side effects.B.javanica,the dried ripe fruit of Brucea javanica(L)Merr(Simaroubaceae),is mainly produced in Guangdong,Guangxi,Hainan and Fujian province of China.In Traditional chinese medicine,B.javanica,bitter in taste and cold in nature,is one of the herbs that can clear heat and remove toxin.It is widely used in the treatment of lung,prostate,and gastrointestinal cancers in clinical practice.Recently,many kinds of anti-tumor active ingredients have been obtained from B.Javanica seeds.However,there are a few reports on the anti-tumor activities of B.javanica peptides.Breast cancer is the primary cause of death in women with tumors.Chinese herbal medicine has obvious advantages and characteristics for the treatment of the disease,and heat-clearing and detoxification is one of the important therapeutic principles.In this study,proteins extracted from B.javanica seeds were selected as the research objects,and the bioactive peptides were obtained by enzymatic hydrolysis.Then ultrafiltration and chromatography were employed to separate and purify the obtained peptides.Finally,peptide faction F9-9 was obtained,which significantly inhibited the proliferation of breast cancer MCF-7 cells.The results were as follows:Analysis on the major component and protein composition of B.javanica seeds,the contents of starch,crude fat,protein,water and ash were dermined by Polarimetry,Soxhlet extraction,Kjeldahl method,Drying method and Muffle furnace respectively,which accounted for 49.26%,16.16%,17.47%,6.74%and 5.61%of the total weight,respectively.Albumin,gulbulin,coxin and glutelin were sequentially extracted and then quantified by Kjeldahl method.The corresponding content of the four kinds of proteins was 15.01%,8.11%,2.47%and 44.92%of the total protein content.Research on preparation of anti-tumor peptides from B.javanica seeds.Three kinds of proteins(albumin,globulin and glutelin)with higher protein content were selected to hydrlyze by pepsin.The hydrolysates were then separated by ultrafiltration centrifuge tube(MWCO = 3 kDa)to collect the ultralitrate.Proliferation inhibition rates on MCF-7 cells of the ultralitrate from the three proteins were 71.12%,75.26%and 14.04%at the concentration of 50?g·mL-1,respectively.As the protein content of globulin was 79.28%,it was selected as the optimal one to obtain anti-tumor peptides.The optimum extraction contditons of globulin were NaCl concentration 5%,extraction time 1.5 h and solid-liquid ratio 1:12 by orthogonal experiments.Finally,different proteases were employed to hydrolyze globulin for 48 h with[S]of 1%and[E]/[S]ratio of 1:10,and pepsin was selected as the optimal protease for hydrolyzing globulin.Separation and purification of anti-tumor peptides.The hydrolysates of B.javanica globulin produced by pepsin were separated and purified by ultrafiltration,SephadexG-10 gel chromatography and RP-HPLC.After being filtered by G4 funnel,the hydrolysates were separated by Millipore ultrafiltration centrifuge tube(MWCO =3 kDa)to obtain the ultrafiltrate with molecular weight less than 3 kDa,which was named BJGH.The result of MTT showed that BJGH had a strong inhibitory effect on the proliferation of MCF-7 cells with the IC50 value of 2.05 ± 0.04?g·mL-1 for 72 h incubation.Nine fractions(designated as F1-F9)were obtained when separating BJGH with Sephadex G-10,and the IC50 value were 33.68±0.30?g·mL-1,69.87±0.04?g·mL-1,32.60 ± 0.04?g·mL-1,2.03 ± 0.04?g·mL-1,2.44 ± 0.02?g·mL-1,0.47±0.02?g·ML-1,0.42±0.03?g·mL-1,0.30±0.02?g·mL-1 and 0.25±0.02?g·ML-1,respectively,after the incubation with MCF-7 cells for 72 h.Among them,fraction F6,F7,F8 and F9 showed significant inhibitory ability in comparison with the control group(P<0.05).Then semi-preparative RP-HPLC was performed to further separate fraction F9,which showed the highest activity,and nine sub-fractions(F9-1-F9-9)were obtained.The anti-tumor activity of the corresponding ones were firstly determined at the concentration of 0.25?g·mL-1.After incubated with MCF-7 cells for 72 h,F9-7,F9-8 and F9-9 showed stronger inhibitory activity compared with other fractions.The corresponding inhibition rates of them were 50.20%,46.91%and 62.74%.The IC50 of F9-7,F9-8 F9-9 were determined to be 0.25 ± 0.01?g·mL-1,0.32±0.01?g·mL-1and 0.124±0.004?g·mL-1 by further studies.Research on anti-tumor mechanism of sub-fraction F9-9.Flow cytometry method was employed to evaluate the effects of fraction F9-9 on cell apoptosis and cell cycle of MCF-7 cells at the concentration of 0.03125?g·mL-1,0.0625?g·mL-1,0.125?g·mL-1.After incubation with MCF-7 cells for 48 h,the results of Annexin V-FITC/PI showed the quantity of apoptotic cells in middle dose group and high dose group were significantly increased compared to the negative control group(P<0.05),except for the low dose group.Briefly,the total quantity of early apoptotic cells increased from 4.47%(control group)to 13.3%and 25.25%for medium and high dose group,respectively,and that of late apoptotic cells increased from 13.40%(control group)to 21.90%and 25.40%.However,the result of PI single staining showed that exposure of MCF-7 cells to low dose of F9-9 led to significant induction of GO/Glgrowth arrest at 48 h(83.79%)compared with the negative control cells(67.34%).Therefore,low dose of F9-9maybe inhibit growth.of MCF-7 cells by blocking cell cycle(G0/G1),while the middle and high dose of F9-9 by inducing cell apoptosis.Then,qRT-PCR was used to detect the mRNA expression of tumor suppressor gene P53 and PTEN,as well as tumor metastasis suppressor gene NM23H-1.The results showed that the mRNA expression of P53,PTEN and NM23H-1 in MCF-7 cells were significantly increased after incubation with F9-9 at the concentration of 0.125?g·mL-1,0.25?g·mL-1,0.5?g·mL-1 for 24 h,compared with the negative control group(P<0.05).Specifically,the mRNA expression of P53 in treated groups of the low,middle and high dose of F9-9 were 1.80,2.02 and 1.84 times as much as that in control group;similarly,the mRNA expression of PTEN and NM23H-1 in the three treated groups were 5.14,3.72 and 4.76 times;and 3.54,2.78 and 2.00 times as much as that in control group,respectively.Therefore,F9-9 may inhibit the proliferation of MCF-7 cells by up-regulating the mRNA expression of the P53,PTEN and NM23H-1.In this study,peptide faction F9-9 with significant inhibitory proliferation on breast cancer cells was obtained from B.javanica gulbulin,which may provide some theoretical guidance for the clinical practice of the traditional medicine. |
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