| Eucommia ulmoides oliver has been used in medicine for more than 2000 years in China.Modern studies have confirmed that the active components of Eucommia ulmoides leaf are similar to those of the bark,and the effects are basically the same,so the leaves can be used as medicine instead of the bark.This dissertation starts from the hypothesis that the Eucommia ulmoides leaf polysaccharides(EULP)with anti-oxidant,anti-inflammatory,immune-enhancing,and anti-breast cancer activities are very likely to have inhibitory activity against HT-29colon cancer cells.Using Eucommia ulmoides leaves harvested in May as materials,the process parameters of ultrasonic and enzymatic-assisted extracting EULP were optimized by the Box-Behnken experimental design;the structural characterization and the in vitro inhibitory activity on HT-29 colon cancer cells of the purified polysaccharides extracted under the optimized conditions were determined;simultaneously,the purified polysaccharide was separated,and the HT-29 colon cancer cells inhibitory activity and molecular structure characteristics of the polysaccharide components of Eucommia ulmoides leaves after separation were studied.Finally,crude polysaccharides were extracted from Eucommia ulmoides leaves harvested in April,June,August,and October,and their contents were determined.The HT-29 colon cancer cells inhibitory activity experiments and the molecular structure analysis were conducted.This article confirms that EULP has colon cancer inhibitory activity,and also provides a scientific and theoretical basis for the development of health foods and medicines using EULP as the main raw material,and guides planters to rationally harvest high-quality Eucommia leaves.1.Optimization of ultrasonic and enzymatic-assisted extraction of EULP.Using Eucommia ulmoides leaves harvested in May as materials,the effects of enzyme complex amount,p H,extraction temperature,ultrasonic power,liquid-solid ratio and extraction time on the yield of polysaccharides were investigated.The Plackett-Burman experiment screened out three factors significantly affecting yield of polysaccharides:p H,ultrasonic power and amount of enzyme complex.The order of significance was as follows:p H>ultrasonic power>amount of enzyme complex.The steepest hill-climbing experiment and the Box-Behnken experiment were carried out on the three significant factors.The optimal conditions of ultrasonic and enzymatic-assisted extraction of EULP were as follows:3.7%of enzyme complex amount,p H 4.0,100 W of ultrasonic power,45 ~oC of extraction temperature,20:1(m L/g)of liquid-solid ratio and 15 min of extraction time.Under the optimized conditions,the actual yield of EULP was 4.79%±0.02%,which was close to the predicted yield of 4.87%and much higher than that by traditional water extraction method.2.Structural characterization and anti-colon cancer activity of EULP.After extracting and purifying the crude polysaccharides from Eucommia ulmoides leaves picked in May by ultrasonic and enzymatic-assisted method,the molecular weight,monosaccharide composition and infrared spectroscopy of the purified EULP were analyzed.The proliferation,migration inhibition and pro-apoptotic activities of EULP on human colon cancer cells were also investigated with HT-29 human colon cancer cells as target.The results showed that the molecular weight of EULP was 1.8×10~5Da,and the polydispersity index Mw/Mn=1.94.The main components of EULP were mannose,galactose and arabinose,which accounted for 13.52%,42.78%and 20.84%of the total polysaccharides,respectively,and it was aβ-type acidic heteropolysaccharide.In the in vitro test of colon cancer inhibitory activity,the inhibition rate of HT-29 colon cancer cell proliferation was 37.45±0.10%after treated with high dose of EULP(750μg/m L)for 72 h,which was higher than that of the positive control group(37.17±0.13%)in the same period.The relative migration rate was 15.89±0.39%,which was much lower than that of blank control group(39.37±0.50%).In the apoptosis analysis experiment,EULP significantly promoted the apoptosis in HT-29 colon cancer cells.3.Isolation of EULP and screening and structural characterization of anti-colon cancer active components.First,EULP was separated and purified by DEAE-52 anion chromatography,and three fractions,EULP-Ⅰ,EULP-Ⅱand EULP-Ⅲ,were obtained.In the colon cancer cells proliferation inhibition experiments,the three fractions all showed inhibitory activity,and EULP-Ⅲhad the highest inhibitory activity.Then,EULP-Ⅲwas further isolated by Sephadex G-100 gel chromatography,and two fractions,EULP-Ⅲ-a and EULP-Ⅲ-b,were obtained.In colon cancer cells proliferation inhibition experiments,EULP-Ⅲ-b had higher inhibition activity than EULP-Ⅲ-a.The structure of EULP-Ⅲ-b was characterized,and the results showed that the molecular weight of EULP-Ⅲ-b was 3.1236×10~4Da,which was much smaller than that before separation.EULP-Ш-b was mainly composed of galactosamine hydrochloride,rhamnose,glucosamine hydrochloride and galactose,the mole ratio of which was0.099:0.394:0.178:0.329.Like the test results before separation,galactose was still the main component of EULP-Ш-b.4.Changes in purity,structural characteristics and anti-colon cancer activity of crude polysaccharides extracted from Eucommia ulmoides leaves at different harvesting periods.Crude polysaccharides were extracted from Eucommia ulmoides leaves harvested in April,June,August and October,which were named EULP1,EULP2,EULP3 and EULP4,respectively.It was found that the polysaccharide contents in EULP1 and EULP4 were higher than those in EULP2 and EULP3.The molecular weights of EULP1,EULP2,EULP3 and EULP4 were 5.837×10~5Da,1.341×10~5Da,7.992×10~4Da and 3.724×10~5Da,respectively.The main components of EULP1 and EULP2 were rhambose and galactose,while the main components of EULP3 and EULP4 were galactose and arabinose.EULP1 and EULP4 had the highest galactose content,reaching 29.1%and 32.9%,respectively.The infrared spectra of the four crude polysaccharides from Eucommia ulmoides leaves were basically the same,and all had the common polysaccharide characteristic peaks.In the anti-colon cancer activity experiment,the proliferation inhibitory activities of EULP1 and EULP4 on HT-29 cells after treatment for 72 h were much higher than those of EULP2 and EULP3.The anti-migration activities on HT-29 cells were in the order of EULP1>EULP4>EULP3>EULP2. |
PDF Full Text Request