| The seed scales of Korean pine are main scales of coniferous trees in Northeast China,but have been rarely used.For the efficient use of forest waste,we want to convert it to have high activity and high value-added products,which has a certain theoretical value and social significance.In this study,the seed scales of Korean pine were selected as the research object,and the polyphenols compounds in it were extracted and separated,as well as the inhibitory effect of polyphenols on tumor activity was studied,the results are as follows:(1)firstly,the seed scales of Korean pine were de-fat used supercritical fluid,and the polyphenols from Korean pine were extracted with ethanol assisted with grind beads,the effect of liquid/material ratio,extraction time,filling percentage of grinding beads,diameter of grinding beads and rotating speed on the yield of polyphenols were studied.On this basis,Plackett-Burman method was used to analyze the factors,and the order of effect were extraction time>liquid/material ration>filling percentage of grinding beads.Using the Box-Behnken response surface optimization process,the best extraction process was as follows:liquid/material ratio was 62:1,extraction time was 2.1h,filling percentage of grinding beads was 62%,under this condition,the yield of polyphenols was 29.09mg/g.(2)Prelimimnary separation and purification of polyphenols crude extracts from Korean pine by macroporous resin.With the static adsorption rate and desorption rate as the index,seven kinds of macroporous adsorption resins were screened,and the AB-8 resin was the best separation medium.Investigate the dynamic elution conditions,and used the orthogonal to optimization the process,the optimal purification process was as follows:70%ethanol as eluent,the dosage of eluent was 4 BV,diameter/length ratio of column was 1:25,elution rate was 0.9Ml/min,the purifity of Korean pine polyphenols was increased from 1 1.56%to 39.82%under above purification conditions.Preliminary identified by HPLC,Korean pine polyphenols containing quercetin(35.86%),p-coumaric acid(16.58%),salicylic acid(12.41%),gallic acid(4.42%);chlorogenic acid(4.07%),catechin(3.25%),rutin(3.18%),phlorizin(3.09%),resveratrol,cinnamic acid,caffeic acid and proanthocyanidins.(3)By silica gel column chromatography,acetic ether,n-butyl alcohol,methanol and water were selected for different polar gradient elution,fractions of Korean pine polyphenols were eluted successively.Take the inhibitory rate of A549 lung adenocarcinoma cell as activity tracing index,the active site was confirmed.12 polyphenols fractions were collected,in the concentration range of 0.2-1.4 mg/mL,through the investigation of inhibition rate of BEAS-2B lung epithelial cell,we confirmed that the isolated part of the 12 polyphenoolic compounds are non-toxicity to normal cells of the body.The most effect of inhibition of A549 tumor cells was n-butanol/methanol fraction,which was significant difference than other fractions(p<0.05).The results of HPLC showed that this fraction contain quercetin(34.14%)and cinnamic acid(31.27%).This fraction showed significant relationship between the dose and effect on the inhibition proliferation of oophoroma Hela,skin cancer A375,colon cancer LoVo and lung adenocarcinoma isolated cells(p<0.05).Single cell gel electrophoresis results showed that this fraction had a persistent effect on DNA of A549 tumor cells within 48h.0.4mg/mL concentration reached the strongest effect when 24h,olive tail moment was 28.14 ±9.64,TDNA reached 59.44%.The results of flow cytometry showed that A549 cells were arrested in GZ/M period by polyphenol compounds.(4)The tumor-bearing mice model was established,with 50,100,200mg/kg d fed Korean pine polyphenols,the results showed that high doses of polyphenols make the inhibition rate was 62.23%,there was no significant difference with the cyclophosphamide on the inhibition effect of tumor.The effects of different doses of Korean oine polyphenols on the immune organ index of the tumor-bearing mice were significantly better than those of the tumor model group and the antitumor drug group.The results of the high sensitivity test(DTH)showed that the high dose of Korean pine polyphenols had a significant inhibitory effect on DTH(p<0.01),and the inhibition effect decreased with decreasing doses,but there was no significant difference(p>0.05).In the inhibition of proliferation of spleen cells,the results showed that both polyphenols and tea polyphenols had a significant effect on the proliferation of spleen cells(p<0.01).The effect of high dose of Korean pine polyphenols on carbon particle clearance was very significant,and the levels of serum TNF-a AND IL-2 in the high dose group were significantly higher than those in the model group(p<0.05).in the study of the mice oxidase system,we found that the activity of T-SOD in the liver,brain and kidney of mice was significantly promoted by the Korean pine polyphenols,and the content of MDA as decreased significantly(p<0.05)at the same time.The effect of high dose of Korean pine polyphenols on the activity of CAT was significantly increased,and the activity of GSH-Px in brain and liver tissue was increased,and there was a significantly difference between the effect and dose.(5)Immunohistochemical method combined with morphological observation of tumor organization was used to study the tumor inhibitory pathway of seed scales of Korean pine polyphenols.Through the morphological observation after HE staining,we can see that the different doses of polyphenols could cause obvious damage of the tumor tissue cells.The results of single cell gel electrophoresis showed that Korean pine polyphenols can continue to function in 72h,showed a dose and time dependence(p<0.05),olive tail moment 35.96 ±6.48,TNDAreached 71.12 ±9.82%,cell cycle arrest in the G2/M period.TUNEL test showed that there was a trend of the dose and effect of the Korean pine polyphenols on the apoptosis of tumor cells.The expression of Caspase-9 and-Caspase 3 in different groups showed a significant consistency(p<0.01),and the expression of Caspase-9 was significantly higher than that of other groups with the high dose polyphenols.The expression of two genes proteins Ptch-1 and Gli-1 of SHH pathway showed a significant correlation,correlation coefficient r=0.945 which mean more significant relation.The expressions of both were inhibition by Korean pine polyphenols,and there was a significant down regulation.Notch-1 and Hes protein in the Notch signaling pathway were both positive,and extremely significant correlation,it showed a dose and effect trend under the different doses of polyphenols.Transcription factor of Hes showed significantly reduced the relationship between the dose and effect.The results indicated that Korean pine polyphenols may be an activity inhibitor of SHH pathway and Notch signaling pathway. |
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