桑树组织中生物碱DNJ的提取及应用研究
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摘要
本论文主要对蚕业资源综合利用中桑枝的综合利用做一定的分析研究。在蚕业综合利用的各项分支中,桑枝的总量大,利用率低,平均每亩桑园年夏伐桑枝可达到500-800kg,按安徽省近50万亩桑园计算,夏伐桑枝的总产量达到2.5×105～4×105吨,桑枝中含有0.1%～0.2%的生物碱1-脱氧野尻霉素(DNJ),DNJ是一种高效抑制人体内α-葡萄糖苷酶活性、可以用于治疗糖尿病的天然产物,价格昂贵。桑枝中按照0.1%左右的提取量提取10%～30%的DNJ粗提物,可生产成品1000～8000吨,作为糖尿病治疗的保健药品可创收数亿元。通过本实验研究,对夏伐桑枝中生物碱DNJ的提取利用和降血糖效果进行了分析,桑枝中DNJ的提取利用可以为桑枝的综合利用提供理论参考。
本实验首先用蒸馏水粗提桑树不同组织中的生物碱1-脱氧野尻霉素(DNJ),通过阳离子交换柱层析对粗提液进行纯化后,用紫外分光光度法测定不同组织中DNJ的含量,并比较不同组织样品中DNJ的含量变化。结果表明:桑树不同组织的样品中DNJ含量有一定差异,桑芽、桑枝和发芽前桑皮中所含DNJ含量较高,桑枝中的含量可达0.15%,发芽前桑皮和桑芽次之,发芽后桑皮、桑叶和桑根中所含DNJ不高,发芽后桑皮所含DNJ只有0.03%,发芽前桑皮中DNJ的含量高于发芽后的桑皮,实验结果为桑树不同组织的提取选择和DNJ的利用提供参考。
将桑树组织中DNJ含量较高的桑枝作为研究对象,选择不同的溶剂和提取条件提取DNJ,比较、分析桑枝中提取DNJ的最佳工艺。对桑枝(主要为夏伐桑枝)的提取工艺研究中,乙醇的提取效果高于蒸馏水,60℃、80%乙醇的条件下提取2小时为DNJ的最佳提取条件,提取率可达到0.176%,对桑枝的三个主要提取条件温度、提取溶液、提取时间进行正交试验,对DNJ提取效果的影响条件中提取温度>提取溶液>提取时间,实验结果为夏伐桑枝中DNJ的提取利用提供了基础。
对不同存放时间的夏伐桑枝进行DNJ的提取实验和分析。结果表明:随着夏伐桑枝存放时间的延长,桑枝中DNJ的含量发生一定的变化,存放半年、存放一年、存放一年半的夏伐桑枝中DNJ的含量分别为0.148%、0.139%、0.126%。
提取、分析桑树不同组织中1-脱氧野尻霉素(DNJ)的含量变化后,参照桑枝中DNJ的含量,通过高血糖小白鼠的动物实验,测定桑枝提取液的降血糖效果,实验结果表明:桑枝提取液的降血糖作用明显,比较不同的小白鼠动物实验组,灌胃等量桑枝提取液、二倍量桑枝提取液、降糖药盐酸二甲双胍片的高血糖小白鼠组血糖值四周后分别下降了7.63%、15.72%、18.41%。同时,在不同存放时间的夏伐桑枝的降血糖作用效果的高血糖小白鼠动物实验中,灌胃存放半年的等量桑枝提取液、存放一年的等量桑枝提取液的高血糖小白鼠组血糖值四周后分别下降了15.83%和6.81%。实验结果为桑枝中DNJ的可提取利用性和夏伐桑枝的存放条件、利用价值提供了参考依据。
The study mainly talks about the utilization of mulberry branches, as a major by-products produced in the sericulture. According to the report, the production of cutting branches can reach to500-800kg/mu for one year and a total output of 2.5×105-4×105 tons was acquired from mulberry gardens of 500 thousand mu in Anhui province. There are 0.1%-0.2% of alkaloids 1-deoxynojirimycins in mulberry branch which used as efficient and expensive diabetes for disease treatment. According to extraction rate of 0.1% DNJ from the tissues, these branches could produce 1000-8000 tons of 10%-30% of DNJ crude extracts and bring out several millions RMB income. In this experiment, extract method and hypoglycemic effect of DNJ from mulberry is analyzed and this study may provide theoretical reference for the further utilization of cutting mulberry branches in summer.
After being extracted by distilled water, DNJ was purified by iron-exchange column chromatography and its content distributed in different tissues was determined by UV spectrophotometric. The results show DNJ content in various parts of mulberry was different: higher levels of DNJ in buds, branches and barks before germinate of mulberry were found, 0.15% in the branches and DNJ contents in barks and buds were followed. However, lower DNJ concentration was determined in leaves and roots of mulberry and was lowest as 0.03% in barks after germinating. These results lay the base for the extraction of DNJ from different tissues of mulberry.
Taken the branches as experimental material, choose the various conditions such as solvents, time and temperature to analyze the technical parameters. By comparison of different extraction conditions, 80% of ethanol, 60℃and 2 hours treament was optimum and the DNJ extraction rate reaches 0.176% ,while extracted with ethanol is better than distilled water. As far as the infuencing factors on DNJ extraction was concerned, the temperature was more than solvent and time.
We also found that with the time increases, the DNJ content in the deposited mulberry branches decreases: 0.148 % for half a year, 0.139% for one year and 0.126% for one and half a year,respectively.
To investigate the blood-glucose lowering effect of DNJ from mulbeery, the animal experiment was performed using hyperglycemic mices model. The results indicates the blood sugar of mice decreases by 7.63%,15.72% and 18.41% after being gavaged with equivalent extract, double amount of extract and metformin, respectively. In addition, blood sugar value of treated mice after 4 weeks decreases with different deposited time of mulberry branches, namely, 15.83% and 6.81% for half a year and one year respectively.
本实验首先用蒸馏水粗提桑树不同组织中的生物碱1-脱氧野尻霉素(DNJ),通过阳离子交换柱层析对粗提液进行纯化后,用紫外分光光度法测定不同组织中DNJ的含量,并比较不同组织样品中DNJ的含量变化。结果表明:桑树不同组织的样品中DNJ含量有一定差异,桑芽、桑枝和发芽前桑皮中所含DNJ含量较高,桑枝中的含量可达0.15%,发芽前桑皮和桑芽次之,发芽后桑皮、桑叶和桑根中所含DNJ不高,发芽后桑皮所含DNJ只有0.03%,发芽前桑皮中DNJ的含量高于发芽后的桑皮,实验结果为桑树不同组织的提取选择和DNJ的利用提供参考。
将桑树组织中DNJ含量较高的桑枝作为研究对象,选择不同的溶剂和提取条件提取DNJ,比较、分析桑枝中提取DNJ的最佳工艺。对桑枝(主要为夏伐桑枝)的提取工艺研究中,乙醇的提取效果高于蒸馏水,60℃、80%乙醇的条件下提取2小时为DNJ的最佳提取条件,提取率可达到0.176%,对桑枝的三个主要提取条件温度、提取溶液、提取时间进行正交试验,对DNJ提取效果的影响条件中提取温度>提取溶液>提取时间,实验结果为夏伐桑枝中DNJ的提取利用提供了基础。
对不同存放时间的夏伐桑枝进行DNJ的提取实验和分析。结果表明:随着夏伐桑枝存放时间的延长,桑枝中DNJ的含量发生一定的变化,存放半年、存放一年、存放一年半的夏伐桑枝中DNJ的含量分别为0.148%、0.139%、0.126%。
提取、分析桑树不同组织中1-脱氧野尻霉素(DNJ)的含量变化后,参照桑枝中DNJ的含量,通过高血糖小白鼠的动物实验,测定桑枝提取液的降血糖效果,实验结果表明:桑枝提取液的降血糖作用明显,比较不同的小白鼠动物实验组,灌胃等量桑枝提取液、二倍量桑枝提取液、降糖药盐酸二甲双胍片的高血糖小白鼠组血糖值四周后分别下降了7.63%、15.72%、18.41%。同时,在不同存放时间的夏伐桑枝的降血糖作用效果的高血糖小白鼠动物实验中,灌胃存放半年的等量桑枝提取液、存放一年的等量桑枝提取液的高血糖小白鼠组血糖值四周后分别下降了15.83%和6.81%。实验结果为桑枝中DNJ的可提取利用性和夏伐桑枝的存放条件、利用价值提供了参考依据。
The study mainly talks about the utilization of mulberry branches, as a major by-products produced in the sericulture. According to the report, the production of cutting branches can reach to500-800kg/mu for one year and a total output of 2.5×105-4×105 tons was acquired from mulberry gardens of 500 thousand mu in Anhui province. There are 0.1%-0.2% of alkaloids 1-deoxynojirimycins in mulberry branch which used as efficient and expensive diabetes for disease treatment. According to extraction rate of 0.1% DNJ from the tissues, these branches could produce 1000-8000 tons of 10%-30% of DNJ crude extracts and bring out several millions RMB income. In this experiment, extract method and hypoglycemic effect of DNJ from mulberry is analyzed and this study may provide theoretical reference for the further utilization of cutting mulberry branches in summer.
After being extracted by distilled water, DNJ was purified by iron-exchange column chromatography and its content distributed in different tissues was determined by UV spectrophotometric. The results show DNJ content in various parts of mulberry was different: higher levels of DNJ in buds, branches and barks before germinate of mulberry were found, 0.15% in the branches and DNJ contents in barks and buds were followed. However, lower DNJ concentration was determined in leaves and roots of mulberry and was lowest as 0.03% in barks after germinating. These results lay the base for the extraction of DNJ from different tissues of mulberry.
Taken the branches as experimental material, choose the various conditions such as solvents, time and temperature to analyze the technical parameters. By comparison of different extraction conditions, 80% of ethanol, 60℃and 2 hours treament was optimum and the DNJ extraction rate reaches 0.176% ,while extracted with ethanol is better than distilled water. As far as the infuencing factors on DNJ extraction was concerned, the temperature was more than solvent and time.
We also found that with the time increases, the DNJ content in the deposited mulberry branches decreases: 0.148 % for half a year, 0.139% for one year and 0.126% for one and half a year,respectively.
To investigate the blood-glucose lowering effect of DNJ from mulbeery, the animal experiment was performed using hyperglycemic mices model. The results indicates the blood sugar of mice decreases by 7.63%,15.72% and 18.41% after being gavaged with equivalent extract, double amount of extract and metformin, respectively. In addition, blood sugar value of treated mice after 4 weeks decreases with different deposited time of mulberry branches, namely, 15.83% and 6.81% for half a year and one year respectively.
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