仙人掌多糖的分离纯化及降血糖的研究
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摘要
本文对单刺仙人掌(Opuntia monacantha Haw.)多糖的分离纯化、糖尿病并发症四条通道的抑制作用及降血糖功能进行了较为系统的研究,研究结果表明:
将响应面分析法应用于仙人掌多糖水浸提工艺的研究是可行的。对多糖浸提得率的影响因素从大到小排序为:水料比>浸提温度>浸提时间。最适浸提条件为:水料比5.5:1,浸提温度75℃,浸提时间2.2h,浸提1次。该条件下仙人掌多糖的浸提得率为0.81%。将超滤膜应用于仙人掌多糖浸提液的纯化和浓缩。结果显示:当选用截留分子质量为10000u的超滤膜,超滤压力为0.20MPa,超滤温度为25℃,料液体积流量控制在0.04～0.05L/min,控制浓缩倍数5～7倍时,粗多糖得率为0.95%。与传统的真空热浓缩法相比,超滤浓缩法的多糖得率相近,但粗多糖中多糖的含量比真空热浓缩法高,所得多糖的品质得到了改善。
将超滤浓缩后的多糖浸提溶液在体系乙醇浓度为55%,4℃下沉降8h,离心后得到粗多糖OP1。上清液再超滤浓缩到粗多糖浓度1.5～2.0%后,加入95%乙醇调节体系乙醇浓度到80%,得沉降粗多糖OP2。另外将超滤浓缩多糖浸提溶液一次性80%乙醇沉降,得粗多糖OP3。OP1、OP2、OP3的多糖沉降率分别为51.72%、45.20%、95.61%。经Sevag法脱蛋白、活性炭脱色后,分别得到初步纯化的仙人掌多糖OPM1、OPM2、OPM3。
通过体外筛选模型,研究了OPM1、OPM2、OPM3对糖尿病并发症机制中四条通路的抑制作用,结果显示:(1)OPM1、OPM2、OPM3均能有效地清除O-2·、·OH。OPM2的作用效果明显强于OPM3及OPM1。在抑制脂质过氧化产物(MDA)试验中,OPM2和OPM3能明显降低大鼠脑组织匀浆中MDA含量,与模型对照组比较,有极显著性差异。(2)在体外蛋白非酶糖化的抑制作用试验中,孵育至第四周时,氨基胍的抑制作用迅速下降,多糖OPM2对AGEs形成抑制作用相对较强,并强于相同剂量的氨基胍。(3)三种多糖对大鼠晶状体中的醛糖还原酶均有不同的抑制作用,并存在一定的量效关系,以OPM2的抑制作用相对较强,其次是OPM3,但都不及阳性对照物依帕司他的作用效果。(4)对蛋白激酶C活性的影响试验中,显示在胞膜组分中,多糖对PKC活性均具有不同程度的抑制,OPM1对PKC的抑制作用要相对强于OPM2和OPM3。
选用抑制糖尿病并发症通道效果较好的OPM2,用于糖尿病小鼠降血糖试验,得出OPM2有一定的降血糖作用,并能改善糖尿病小鼠消瘦、多饮的症状,但降血糖作用不及阳性对照药物二甲双胍明显;同时糖尿病小鼠血清高密度脂蛋白胆固醇含量增加、总胆固醇含量降低,说明多糖具有调节血脂的作用。
OPM2先经DEAE Sepharose CL-6B柱层析分级,得一主要多糖组分OPMH1。接着用Sephadex G-100葡聚糖凝胶柱对多糖OPMH1进一步层析,得两个多糖组分OPMH1Ⅰ和OPMH1Ⅱ,经纯度鉴定,OPMH1Ⅰ和OPMH1Ⅱ都为均一性多糖分子。采用凝胶渗透色谱(GPC)法、间羟基联苯法和气相色谱(GC)法分别对OPMH1Ⅰ和OPMH1Ⅱ分子进行测定,得出:OPMH1Ⅰ重均分子量Mw为42865Da,单糖组成为鼠李糖、阿拉伯糖、葡萄糖、葡萄糖醛酸,摩尔比为9.15:1.00:6.84:0.51;OPMH1Ⅱ重均分子量Mw为20669Da,单糖组成为鼠李糖、甘露糖、葡萄糖、葡萄糖醛酸,摩尔比为8.72:1.00:6.19:1.06。
经高碘酸氧化及Smith降解,结合红外光谱(IR)和核磁共振(NMR)图谱分析,显示OPMH1Ⅰ以鼠李糖为主链,以1→2和1→3键连接状态存在,二者的摩尔百分比为67.3%和32.7%;侧链连接有葡萄糖和阿拉伯糖,这两种糖均以1→3键连接。OPMH1Ⅱ以鼠李糖为主链,以1→2和1→3键连接状态存在,二者的摩尔百分比为60.9%和39.1%;侧链连接葡萄糖和甘露糖,这两种糖均以1→3键连接。
The isolation, purification and structure characterization of polysaccharides of Opuntia monacantha cladode (POMC), as well as their inhibitory effects on the four factors in resulting diabetic chronic complication and the control of blood glucose were investigated in this study. The main contents and results are as follows:
Aiming at optimization of the extraction of POMC, response surface methodology was adopted. The results showed that the optimum conditions of Opuntia polysaccharides extraction were as follows: ratio of water to material of 5.5:1, extraction temperature of 75℃, extraction time of 2.2h. Under these conditions the extraction yield could be up to 0.81% by one-time extraction.
The purification and concentration of POMC were carried out via membrane ultrafitration using the membranes with different molecular weight cut-off. The optimum conditions for the ultrafiltration were finally determined. It was found that, for the membrane with a molecular mass cut-off of 10000u, when the pressure, temperature, purification time, flow rate and concentration multiple were 0.20 MPa, 25℃, 35 min, 0.04-0.05 L/min and 5~7, respectively, a POMC yield of 0.95% was reached. Moreover, as compared with the traditional vacuum concentration, membrane ultrafiltration was easier to obtain high-quality crude POMC with a higher content of polysaccharide, which was helpful for the further extraction and purification of polysaccharides in the following process.
After concentration by membrane ultrafiltration, OP1 was obtained by precipitation extracts with 55% ethanol solution. The supernatant was added ethanol to final concentration of 80% to precipitate OP2. OP3 was obtained by precipitation extracts with 80% of ethanol solution directly. Then, OPM1、OPM2、OPM3 were gained after purification.
In vitro trials of inhibitory effects on the four indices reflecting the resulting diabetic chronic complication were evaluated. The results showed: 1) polysaccharide OPM1、OPM2、OPM3 had significant free-radical scavenging activity. Amongst, OPM2 had the highest free-radical scavenging activity. OPM2 and OPM3 had inhibitory effects on Malondialdehyde to different extent; 2) OP2 showed stronger inhibition effect on AGE than aminoguanidine with the same dose at fourth week; 3) OPM2 also showed the strongest inhibition effect on aldose reductase (AR) activity, but lower than epalrestat, the positive control; 4) OPM1 showed the strongest inhibition effect on protein kinase C (PKC) activity in the extraction from cell membrane.
OPM2 which had a good performance on above four indices was chosen for hypoglycemic analysis. The resutls showed that it had significant hypoglycemic effect, but lower than aminiguanidine. The body weight loss and polydipsia of diabetic rats were decreased. The high density lipoprotein level and total cholesteral level were increased and decreased, respectively.
Then OPM2 was chromatographed on a DEAE Sepharose CL-6B anion exchange column to yield a major fraction (OPMH1). OPMH1 was subjected to further purification on a Sephadex G-100 gel filtration column. Two major fractions, OPMH1 I和OPMH1 II, were collected. By analyses of gel permeation chromatography (GPC), meta-hydroxydiphenyl method and gas chromatography (GC), OPMH1 I, which had a average molecular weight of 42.87 kDa, comprised mainly of rhamnose, arabinose, glucose and glucuronic acid in the molar ratio of 9.15: 1.00: 6.84: 0.51. While OPMH1 II, which had an average molecular weight of 20.67 kDa, comprised mainly of rhamnose, mannose, glucose and glucuronic acid in the molar ratio of 8.72: 1.00: 6.19: 1.06.
The results of periodate oxidation and Smith degradation showed that the backbone of OPMH1 I was 1→2 rhamnose and 1→3 rhamnose. Their molar ratio was 2.06: 1. The side chains mainly comprised of branched (1→3)-linked glucose and (1→3)-linked arabinose. The backbone of OPMH1 II was 1→2 rhamnose and 1→3 rhamnose. Their molar ratio was 1.56: 1. The side chains mainly comprised of branched (1→3)-linked glucose and (1→3)-linked mannose.
将响应面分析法应用于仙人掌多糖水浸提工艺的研究是可行的。对多糖浸提得率的影响因素从大到小排序为:水料比>浸提温度>浸提时间。最适浸提条件为:水料比5.5:1,浸提温度75℃,浸提时间2.2h,浸提1次。该条件下仙人掌多糖的浸提得率为0.81%。将超滤膜应用于仙人掌多糖浸提液的纯化和浓缩。结果显示:当选用截留分子质量为10000u的超滤膜,超滤压力为0.20MPa,超滤温度为25℃,料液体积流量控制在0.04～0.05L/min,控制浓缩倍数5～7倍时,粗多糖得率为0.95%。与传统的真空热浓缩法相比,超滤浓缩法的多糖得率相近,但粗多糖中多糖的含量比真空热浓缩法高,所得多糖的品质得到了改善。
将超滤浓缩后的多糖浸提溶液在体系乙醇浓度为55%,4℃下沉降8h,离心后得到粗多糖OP1。上清液再超滤浓缩到粗多糖浓度1.5～2.0%后,加入95%乙醇调节体系乙醇浓度到80%,得沉降粗多糖OP2。另外将超滤浓缩多糖浸提溶液一次性80%乙醇沉降,得粗多糖OP3。OP1、OP2、OP3的多糖沉降率分别为51.72%、45.20%、95.61%。经Sevag法脱蛋白、活性炭脱色后,分别得到初步纯化的仙人掌多糖OPM1、OPM2、OPM3。
通过体外筛选模型,研究了OPM1、OPM2、OPM3对糖尿病并发症机制中四条通路的抑制作用,结果显示:(1)OPM1、OPM2、OPM3均能有效地清除O-2·、·OH。OPM2的作用效果明显强于OPM3及OPM1。在抑制脂质过氧化产物(MDA)试验中,OPM2和OPM3能明显降低大鼠脑组织匀浆中MDA含量,与模型对照组比较,有极显著性差异。(2)在体外蛋白非酶糖化的抑制作用试验中,孵育至第四周时,氨基胍的抑制作用迅速下降,多糖OPM2对AGEs形成抑制作用相对较强,并强于相同剂量的氨基胍。(3)三种多糖对大鼠晶状体中的醛糖还原酶均有不同的抑制作用,并存在一定的量效关系,以OPM2的抑制作用相对较强,其次是OPM3,但都不及阳性对照物依帕司他的作用效果。(4)对蛋白激酶C活性的影响试验中,显示在胞膜组分中,多糖对PKC活性均具有不同程度的抑制,OPM1对PKC的抑制作用要相对强于OPM2和OPM3。
选用抑制糖尿病并发症通道效果较好的OPM2,用于糖尿病小鼠降血糖试验,得出OPM2有一定的降血糖作用,并能改善糖尿病小鼠消瘦、多饮的症状,但降血糖作用不及阳性对照药物二甲双胍明显;同时糖尿病小鼠血清高密度脂蛋白胆固醇含量增加、总胆固醇含量降低,说明多糖具有调节血脂的作用。
OPM2先经DEAE Sepharose CL-6B柱层析分级,得一主要多糖组分OPMH1。接着用Sephadex G-100葡聚糖凝胶柱对多糖OPMH1进一步层析,得两个多糖组分OPMH1Ⅰ和OPMH1Ⅱ,经纯度鉴定,OPMH1Ⅰ和OPMH1Ⅱ都为均一性多糖分子。采用凝胶渗透色谱(GPC)法、间羟基联苯法和气相色谱(GC)法分别对OPMH1Ⅰ和OPMH1Ⅱ分子进行测定,得出:OPMH1Ⅰ重均分子量Mw为42865Da,单糖组成为鼠李糖、阿拉伯糖、葡萄糖、葡萄糖醛酸,摩尔比为9.15:1.00:6.84:0.51;OPMH1Ⅱ重均分子量Mw为20669Da,单糖组成为鼠李糖、甘露糖、葡萄糖、葡萄糖醛酸,摩尔比为8.72:1.00:6.19:1.06。
经高碘酸氧化及Smith降解,结合红外光谱(IR)和核磁共振(NMR)图谱分析,显示OPMH1Ⅰ以鼠李糖为主链,以1→2和1→3键连接状态存在,二者的摩尔百分比为67.3%和32.7%;侧链连接有葡萄糖和阿拉伯糖,这两种糖均以1→3键连接。OPMH1Ⅱ以鼠李糖为主链,以1→2和1→3键连接状态存在,二者的摩尔百分比为60.9%和39.1%;侧链连接葡萄糖和甘露糖,这两种糖均以1→3键连接。
The isolation, purification and structure characterization of polysaccharides of Opuntia monacantha cladode (POMC), as well as their inhibitory effects on the four factors in resulting diabetic chronic complication and the control of blood glucose were investigated in this study. The main contents and results are as follows:
Aiming at optimization of the extraction of POMC, response surface methodology was adopted. The results showed that the optimum conditions of Opuntia polysaccharides extraction were as follows: ratio of water to material of 5.5:1, extraction temperature of 75℃, extraction time of 2.2h. Under these conditions the extraction yield could be up to 0.81% by one-time extraction.
The purification and concentration of POMC were carried out via membrane ultrafitration using the membranes with different molecular weight cut-off. The optimum conditions for the ultrafiltration were finally determined. It was found that, for the membrane with a molecular mass cut-off of 10000u, when the pressure, temperature, purification time, flow rate and concentration multiple were 0.20 MPa, 25℃, 35 min, 0.04-0.05 L/min and 5~7, respectively, a POMC yield of 0.95% was reached. Moreover, as compared with the traditional vacuum concentration, membrane ultrafiltration was easier to obtain high-quality crude POMC with a higher content of polysaccharide, which was helpful for the further extraction and purification of polysaccharides in the following process.
After concentration by membrane ultrafiltration, OP1 was obtained by precipitation extracts with 55% ethanol solution. The supernatant was added ethanol to final concentration of 80% to precipitate OP2. OP3 was obtained by precipitation extracts with 80% of ethanol solution directly. Then, OPM1、OPM2、OPM3 were gained after purification.
In vitro trials of inhibitory effects on the four indices reflecting the resulting diabetic chronic complication were evaluated. The results showed: 1) polysaccharide OPM1、OPM2、OPM3 had significant free-radical scavenging activity. Amongst, OPM2 had the highest free-radical scavenging activity. OPM2 and OPM3 had inhibitory effects on Malondialdehyde to different extent; 2) OP2 showed stronger inhibition effect on AGE than aminoguanidine with the same dose at fourth week; 3) OPM2 also showed the strongest inhibition effect on aldose reductase (AR) activity, but lower than epalrestat, the positive control; 4) OPM1 showed the strongest inhibition effect on protein kinase C (PKC) activity in the extraction from cell membrane.
OPM2 which had a good performance on above four indices was chosen for hypoglycemic analysis. The resutls showed that it had significant hypoglycemic effect, but lower than aminiguanidine. The body weight loss and polydipsia of diabetic rats were decreased. The high density lipoprotein level and total cholesteral level were increased and decreased, respectively.
Then OPM2 was chromatographed on a DEAE Sepharose CL-6B anion exchange column to yield a major fraction (OPMH1). OPMH1 was subjected to further purification on a Sephadex G-100 gel filtration column. Two major fractions, OPMH1 I和OPMH1 II, were collected. By analyses of gel permeation chromatography (GPC), meta-hydroxydiphenyl method and gas chromatography (GC), OPMH1 I, which had a average molecular weight of 42.87 kDa, comprised mainly of rhamnose, arabinose, glucose and glucuronic acid in the molar ratio of 9.15: 1.00: 6.84: 0.51. While OPMH1 II, which had an average molecular weight of 20.67 kDa, comprised mainly of rhamnose, mannose, glucose and glucuronic acid in the molar ratio of 8.72: 1.00: 6.19: 1.06.
The results of periodate oxidation and Smith degradation showed that the backbone of OPMH1 I was 1→2 rhamnose and 1→3 rhamnose. Their molar ratio was 2.06: 1. The side chains mainly comprised of branched (1→3)-linked glucose and (1→3)-linked arabinose. The backbone of OPMH1 II was 1→2 rhamnose and 1→3 rhamnose. Their molar ratio was 1.56: 1. The side chains mainly comprised of branched (1→3)-linked glucose and (1→3)-linked mannose.
引文
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