CN115414379A - 三七多糖sqp20在制备治疗肠损伤与炎性浸润的药物的应用 - Google Patents
三七多糖sqp20在制备治疗肠损伤与炎性浸润的药物的应用 Download PDFInfo
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- CN115414379A CN115414379A CN202210987373.7A CN202210987373A CN115414379A CN 115414379 A CN115414379 A CN 115414379A CN 202210987373 A CN202210987373 A CN 202210987373A CN 115414379 A CN115414379 A CN 115414379A
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- sqp20
- polysaccharide
- notoginseng
- intestinal injury
- lps
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Abstract
本发明公开了三七多糖SQP20在制备治疗肠损伤与炎性浸润的药物的应用，所述三七多糖SQP20的分子量为4.84×105Da，单糖组成为甘露糖、鼠李糖、葡萄糖和半乳糖，摩尔比为3.78:1.89:75.81:18.52。通过GC‑MS和核磁结果分析得到其结构为具有少量Glcp‑(1→、→4)‑Manp‑(1→、→3)‑Rhap‑(1→作为侧链链接在葡萄糖半乳聚糖的(1→4)‑Glcp的O‑6上。所述三七多糖SQP20是从三七粉中提取分离出新的三七多糖SQP20，经过试验证明三七多糖SQP20能够改善LPS和束缚应激小鼠肠损伤以及炎性浸润，有利于资源的充分利用。
Description
技术领域
本发明涉及三七多糖，尤其涉及三七多糖SQP20在制备治疗肠损伤与炎性浸润的药物的应用。
背景技术
三七又称为田七，是五加科草本植物三七Panaxnoyoginseng(Burk.)F.H.Chen的干燥根和根茎，甘、微苦，温，道地产地是云南文山。归心、肝、脾经，被广泛用于临床。已有研究发现中药三七具有消肿止痛、化瘀止血、抗炎及保肝利胆作用，常用于治疗跌打损伤、外伤止血、出血性瘀滞、便血及吐血等疾病。现有报道指出三七的有效成分包括多糖、皂苷、黄酮类、炔醇类以及挥发油等。
多糖是由多个单糖分子脱水缩合而成的一类结构复杂的糖类物质，结构单位之间以糖苷键相连接，常见的糖苷键有ɑ-1,3糖苷键、β-1,6糖苷键、β-1,4糖苷键、ɑ-1,4糖苷键和β-1,3糖苷键等。多糖的极性极大，易溶于水，因此常用热水提取法、酸碱浸提法、酶提取法、微波辅助提取法、超声辅助提取法等。众多报道指出植物多糖具有调节肠道微生态、抗氧化、降血糖、降血脂、免疫调节、抗肿瘤等多种生物活性，还具有来源广泛、安全无毒、活性高、环境亲和力高等优点，在食品及生物医药领域具有广阔的应用前景。因此，深入研究三七中多糖成分，具有良好的应用前景。
目前随着研究不断深入，中药三七中多糖总量约为9.45％，并且发现三七多糖在抗肿瘤、抗衰老、降低血糖、保护生殖系统、免疫调节、保肝、抗氧化能力等方面都有疗效。因此，三七多糖在将来会在各个疾病的防治中均可发挥重要作用。然而，与三七其他有效成分相比，三七多糖的研究并不多，原因可能是其在提取、分离及其他药理方面的研究远不及其他植物多糖透彻。因此，针对三七多糖的成分进一步研究，具有广阔的应用前景。
发明内容
本发明的目的在于提供三七多糖SQP20在制备治疗肠损伤与炎性浸润的药物的应用。
具体地，三七多糖SQP20在制备治疗肠损伤与炎性浸润的药物的应用，所述三七多糖SQP20的分子量为4.84×105Da，单糖组成为甘露糖、鼠李糖、葡萄糖和半乳糖，摩尔比为3.78:1.89:75.81:18.52，具有Glcp-(1→、→4)-Manp-(1→、→3)-Rhap-(1→作为侧链链接在葡萄糖半乳聚糖的(1→4)-Glcp的O-6上。
本发明所述三七多糖SQP20由以下方法提取而得：取三七粉按固液比为1﹕10g/mL加入蒸馏水在80℃下提取3次，每次提取2h，合并3次提取液，在50℃的减压下在旋转蒸发器中浓缩到约5L，用AB-8大孔树脂脱色，用Sevag法脱蛋白，然后进行浓缩，在浓缩液中加入乙醇至乙醇浓度为20％，静置沉降，过滤获得沉淀物，即为三七多糖SQP20。经过分级醇沉和DEAE柱层析，从三七植物中分离得到一种分子量为4.84×105Da的均一多糖，命名为三七多糖SQ20。PMP柱前衍生实验表明，三七多糖SQ20是由甘露糖、鼠李糖、葡萄糖和半乳糖组成的，单糖之前比例为3.78:1.89:75.81:18.52。通过GC-MS、核磁和甲基化结果分析得到其结构为具有少量Glcp-(1→、→4)-Manp-(1→、→3)-Rhap-(1→作为侧链链接在葡萄糖半乳聚糖的(1→4)-Glcp的O-6上。
本发明从三七粉中提取分离出新的三七多糖SQP20，经过试验证明三七多糖SQP20能够改善LPS和束缚应激小鼠肠损伤以及炎性浸润。
附图说明
图1是三七多糖的GPC图谱。
图2是混合单糖标准品(A)和SQP20(B)经PMP衍生化后的高效液相色谱图。
图3是发现4-连接吡喃葡萄糖基残基
1,4,5-Tri-Oacetyl-1-deuterio-2,3,6-tri-O-methyl-glucitol。
图4是发现4-连接吡喃半乳糖基残基
1,4,5-Tri-O-acetyl-1-deuterio-2,3,6-tri-O-methyl-galactitol。
图5是发现终端吡喃葡萄糖基残基
1,5-Di-O-acetyl-1-deuterio-2,3,4,6-tetra-O-methyl-glucitol。
图6是发现4,6-连接吡喃葡萄糖基残基
1,4,5,6-Tetra-O-acetyl-1-deuterio-2,3-di-O-methyl-glucitol。
图7是发现4-连接甘露吡喃糖基残基
1,4,5-Tri-O-acetyl-1-deuterio-2,3,6-tri-O-methyl-mannitol。
图8是发现3-连接-6-脱氧甘露吡喃糖基(Rha)残基
1,3,5-Tri-O-acetyl-1-deuterio-6-deoxy-2,4-di-O-methyl-mannitol。
图9-1至图9-5是多糖的核磁共振波图谱；SQP20 1H NMR(A),13C NMR(B),1H-1HCOSY(C),1H-13C HSQC(D),1H-13C HMBC(E)。
图10是三七多糖SQP20的可能结构单元。
图11是三七多糖SQP20可减轻内毒素和束缚应激小鼠的肠道损伤和炎性细胞浸润，n＝3；
A是染色组织切片图；B是条形图。
图12是三七多糖SQP20降低内毒素和束缚应激诱导的小鼠肠道NLRP3和肿瘤坏死因子-α的表达，n＝3。
图13是三七多糖SQP20改善内毒素和束缚应激小鼠肠道NF-κB的表达，n＝3；免疫荧光染色图像用NF-κB标记，绿色表示NF-κB，蓝色表示DAPI；比例尺＝100μm。
图14是三七多糖SQP20可改善内毒素和束缚应激小鼠肠道中超氧化物歧化酶的表达。
采用免疫组织化学和条形图方法对肠组织进行检测。超氧化物歧化酶阳性表达代表图和柱状图。(n＝3)，标尺＝100μm。和正常小鼠比较，*P<0.05，**P<0.01，和LPS+RS组比较，#P<0.05。
具体实施方式
以下列举具体实施例对本发明进行说明。需要指出的是，实施例只用于对本发明作进一步说明，不代表本发明的保护范围，其他人根据本发明做出的非本质的修改和调整，仍属于本发明的保护范围。
1实验材料
1.1实验动物
BALB/c小鼠(SPF级)购买自广东省医学实验动物中心，动物许可证为SCXK(Guangdong,2018-0002)。
1.2实验试剂
1.3实验仪器
2三七多糖的提取分离鉴定
2.1三七多糖的提取
参考文献报道的方法对重量为5.0kg的三七进行多糖提取。三七样品用固液比为1﹕10g/mL的蒸馏水在80℃下提取3次，每次提取2h，合并3次提取液，在50℃的减压下在旋转蒸发器中浓缩到约5L，加入无水乙醇使最终乙醇浓度逐步调整为20％、40％、60％、80％(v/v)来依次获得沉淀物。沉淀离心，冻干后分别用去离子水溶解和透析，透析72h(1000Da拦截)，冷冻干燥分别得到SQP20、SQP40、SQP60和SQP80四个多糖组分。用多糖粉(WSQPs)干重与原料初始重量的比值(WSample)来确定SQPs的萃取率：得率(％)＝WSQPs/WSample×100％。
2.2三七多糖的化学组成和单糖组成分析
以D-葡萄糖为标准品，采用苯酚-硫酸法测定中性碳水化合物含量。糖醛酸含量按间羟基二苯基硫酸测定，以半乳糖醛酸作标准。以牛血清白蛋白(BSA)为标准，采用Bradford‘s法测定蛋白质含量。采用1-苯基-3-甲基-5-吡唑啉酮(PMP)柱前衍生化高效液相色谱法测定SQP20的单糖组成。简而言之，10mg的多糖样品用3mL浓度为4M的三氟乙烷在110℃下水解4h，残渣用甲醇洗涤，多次冷冻干燥，直到溶液在真空下浓缩后完全脱除三聚氰胺。残渣在蒸馏水中重新溶解，加入PMP甲醇溶液和NaOH溶液，并以葡萄糖、半乳糖、鼠李糖、甘露糖、葡萄糖醛酸、半乳糖醛酸、木糖、阿拉伯糖和岩藻糖为单糖标准进行衍生化。混合物用盐酸中和。然后加入氯仿，提取成三份，去除有机相。采用COSMOSIL5C18-PAQ色谱柱(4.6×150mm，5μm)，以0.05M磷酸二氢钾(pH＝6.9)-乙腈(体积比80﹕20)为流动相，1.0mLmin-1洗脱。
2.3三七多糖分子量分析
采用高效毛细管气相色谱(HPGPC)三柱(Waters Ultra Water Gele250、1000和2000；30cm×7.8mm；6μm颗粒)测定SNP的相对分子质量。用T系列葡聚糖标准品绘制了测定SQP20相对分子质量的标准曲线。Log(MW)与洗脱时间(T)的校正曲线为：log(MW)＝-0.1841T+12.1568，R2＝0.9843。
2.4三七多糖的甲基化分析
为了确定糖基键，SQP20按照Hakomori的方法进行了甲基化，并进行了适当的修改。具体地，阿拉伯半乳聚糖SQP20(10.0mg)是在干燥条件下用P2O5进行的。在氮气气氛下，向无水脱气的二甲基亚砜(DMSO)中滴加1.5m L甲基亚磺基亚甲基钠(SMSM)，在室温下搅拌过夜。用超声波法在冰浴下滴加1.5毫升的碘甲烷。采用蒸馏水透析和冷冻干燥的方法收集甲基化多糖。重复上述步骤，直到完成甲基化，经FT-IR光谱证实。没有OH-1振动(3000-3500cm-1)表明完全甲基化。预甲基化后的多糖在100℃下用98％的甲酸4mL水解6h，加入甲醇，蒸发干燥3次，除去多余的甲酸。然后将样品溶解，在110℃下用5mL浓度为2M的TFA水解2h，在室温下冷却后，加入甲醇，蒸发至干燥，去除多余的TFA。将残渣溶解在3mL蒸馏水中，加入30mg NaBH4还原，用25％的HOAc中和，直到停止生成气体。旋转干燥后的样品在110℃下用乙酸酐乙酰化1h，然后用氯仿-水体系提取三次，收集氯甲烷相，得到甲基化的乙酸阿迪醇，并用GC-MS进行分析。
2.5三七多糖核磁共振分析
所有样品(30.0mg)在D2O中溶解，并多次冷冻干燥，以便将H质子完全交换到氘中。随后，将样品在D2O中溶解过夜，然后进行核磁共振分析。用Avance-600型核磁共振波谱仪记录光谱。
3三七多糖对束缚应激诱导下LPS诱导肠炎模型的保护作用
3.1实验方法
小鼠口服三七多糖150mg/kg，1次/天，连续7天。第5天给予束缚应激(RS)1次，连续18h，第6天给予小剂量脂多糖(15μg/kg)腹腔注射诱导肠损伤。末次给药后1h，在乙醚麻醉下解剖小鼠，收集肠道组织，以-80℃保存。
肠组织在4％多聚甲醛固定3天后，取肠组织进行石蜡包埋，制备4μm厚度肠组织切片。切片用苏木精-伊红染色(HE染色)，用Pannorama MIDI扫描显微镜(3D HISTECH)观察，用Case Viewer软件分析。
肠道组织切片脱蜡至水份，在微波炉中与柠檬酸-钠反应5min。用过氧化氢室温孵育10min，用PBS洗涤3次。然后与山羊血清室温孵育15min，加入一抗NLRP3(1﹕50，Abcam)、TNF-α(1﹕500，Santa Cruz)和超氧化物歧化酶(1﹕250，中杉)，室温孵育2小时，洗涤3次，与二抗在37℃孵育15min。随后，在相同温度下，加入辣根过氧化物酶15min。切片用二氨基联苯胺(DAB)显色，蒸馏水洗涤，复染，脱水，中性胶封，显微镜下观察。用Image-J软件对图像进行分析。
如上所述，将肠组织切片脱蜡至混浊，并回收抗原。切片用0.1％Triton X-100和0.1M盐酸渗透10min，然后用PBS清洗3次。用过氧化氢在室温孵育15min，用PBS洗涤3次。然后用山羊血清在室温下封闭1h，加入一抗NF-κB(1﹕400，Abcam)，在4℃孵育过夜，然后洗涤3次，再与Alexa-Folor二抗孵育1h。4’，6-二氨基-2-苯基吲哚(DAPI)用于检测远离光线的细胞核，孵育10min。用抗荧光猝灭固定剂(公司)固定切片，在显微镜下观察。
4实验结果
4.1三七多糖得率、化学组成和单糖组成
表1展示了SQP20、SQP40、SQP60和SQP80的产量，其中SQP80的产量显著高于其他三种(p<0.05)。
表1 SQP20,SQP40,SQP60 and SQP80萃取率和化学成分
4.2三七多糖SQP20的分子量及单糖组成分析
根据葡聚糖标准品的色谱图，拟合重均相对分子质量(MW，Da)和洗脱体积(V，mL)，并计算标准曲线，Log(MW)＝-0.1236T+10.37。根据标准曲线计算出SQP20的重均相对分子质量为4.84×105Da。如图1所示，三七多糖SQP20的GPC图谱显示单一对称的色谱峰，表明SQP20是一种均一的多糖。
如图2所示，经三氟乙酸水解液和PMP衍生化后，用高效液相色谱法测定了三七多糖SQP20的单糖组成。三七多糖SQP20由甘露糖、鼠李糖、葡萄糖和半乳糖组成，摩尔比为3.78:1.89:75.81:18.52。
4.3三七多糖SQP20基因甲基化分析
根据单糖组成结果，三七多糖SQP20不含糖醛酸。因此采用中性多糖的甲基化方法进行检测，甲基化结果如表1所示。多糖SQP20主要由6种糖基组成。其组成由编码甲基化的糖质量片段(m/z)面积比确定连接类型级质谱，分别为→4)-Glcp-(1→(58.22％,图3),→ 4)-Galp-(1→(20.38％,图4),Glcp-(1→(9.83％,图5),→4,6)-Glcp-(1→(9.17％,图6), →4)-Manp-(1→(1.61％,图7)→3)-Rhap-(1→(0.79％,图8)。综上所述，三七多糖SQP20的主链为葡萄糖半乳聚糖，与单糖组成一致。甲基化结果表明，三七多糖SQP20中含有少量的Glcp-(1→、→4)-Manp-(1→、→3)-Rhap-(1→作为支链链接在(1→4)-Galp残基的O-6上，参见图3-8。
由图9-1A可知，三七多糖SQP20的1H NMR谱图包含了2个主要的异头氢信号，分别为δ＝5.32和δ＝4.99，根据甲基化和单糖组成结果，将这两个异头氢归属为葡萄糖和半乳糖的异头氢，由此可以推断出多糖SQP20中的葡萄糖和半乳糖为α构型。在δ＝1.28和δ＝1.09处信号表明多糖SQP20中含有少量的鼠李糖残基。由图9-2B可知，多糖SQP20的13C NMR谱图包含了4个主要的异头碳信号，分别为δ＝101.44、δ＝99.67、δ＝95.81和91.91，结合甲基化和单糖组成结果，将这四个异头碳信号分别归属为半乳糖、葡萄糖、甘露糖和鼠李糖的异头碳信号。在甲基化结果表明糖残基A和糖残基B在多糖SQP20中占比将近80％，而糖残基C、糖残基D和糖残基E在多糖SQP20中占比均未超过10％，所以在二维核磁共振谱图(图9-2B和9-3C)中只归属了糖残基A和糖残基B的信息(表2)，未归属糖残基C、糖残基D和糖残基E的信息。在HMBC谱图(图9-5E)中，显示出糖残基A-H1和糖残基A-C4交叉信号，糖残基A-H4和糖残基A-C1交叉信号，说明多糖SQP20主要的链接方式为→4)-Glcp-(1→4)-Glcp-(1→。糖残基B-H1和糖残基A-C4交叉信号说明多糖SQP20中存在→4)-Galp-(1→4)-Glcp-(1→。甲基化结果表明多糖SQP20中存在10％的Glcp-(1→6)-Glcp-(1→分枝，通过GC-MS、核磁和甲基化结果分析得到其结构为具有少量Glcp-(1→、→4)-Manp-(1→、→3)-Rhap-(1→作为侧链链接在葡萄糖半乳聚糖的(1→4)-Glcp的O-6上。
表2.SQP20的1H NMR and 13C NMR图谱分布
5三七多糖SQP20改善LPS和束缚应激小鼠肠损伤以及炎性浸润
与正常小鼠(CON组)相比，RS组、LPS组和LPS+RS组大鼠肠组织肌层厚度明显减少，炎性细胞浸润明显减少，并伴有肠绒毛破坏(如图11所示)。同时，炎症小体NLRP3和TNF-α的阳性表达增加(如图12所示)。注射LPS的小鼠在给予三七多糖SQP20治疗后在炎症、肠道绒毛和肌层厚度方面与LPS组比较有改善的趋势，但没有显著性差异，但与LPS+RS组比较具有显著的改善作用。NLRP3有改善的趋势但没有显著性差异，TNF-α的表达也无显著差异。但LPS+RS刺激下，三七多糖SQP20治疗后能显著减少炎性细胞数，减少肠组织中NLRP3和TNF-α的阳性表达，恢复肠绒毛形态和肌层厚度。免疫荧光结果还显示，与CON组相比，LPS+RS组大鼠肠组织中的肠肌和绒毛组织中NF-κB的表达明显增强，注射三七多糖SQP20后表达不明显(如图13所示)。以上结果说明三七多糖SQP20能显著改善LPS和束缚应激诱导小鼠肠损伤以及炎性浸润。
肠组织进行H&E染色和条形图分析，图11展示了代表性图表(A)和柱状图(B)。黑色箭头表示肌层，蓝色箭头表示肠道绒毛，红色箭头表示炎性细胞浸润。和模型组比较，**P<0.01，和LPS组比较，$P<0.05，和LPS+RS组比较，##P<0.01。CON代表正常小鼠，LPS代表内毒素处理的小鼠，RS代表束缚应激处理的小鼠，而LPS+RS代表内毒素和束缚应激处理的小鼠。LPS+SQP20和LPS+RS+SQP20分别代表内毒素处理的小鼠或给予SQP20 7天的内毒素和束缚应激处理的小鼠。
采用免疫组织化学和条形图方法对肠组织进行检测，结果见图12所示的NLRP3(A)、TNF-α(B)阳性表达代表图和柱状图。标尺＝100μm。和正常小鼠比较，*P<0.05，**P<0.01，和LPS组比较，$$P<0.01，和RS组比较，&&P<0.01，和LPS+RS组比较，##P<0.01。
6三七多糖SQP20对内毒素和束缚应激所致小鼠氧化应激的影响
如图14所示，与CON组比较，RS组、LPS组和LPS+RS组小鼠肠组织中SOD的阳性表达减少。注射SQP20的LPS组大鼠的超氧化物歧化酶表达有上调的趋势但是无显著性差异。而SQP20能显著增加LPS和RS刺激下小鼠肠道组织中SOD的阳性表达。
以上列详细说明是针对本发明的生产实施例的举例说明，但该实施例并非用以限制本发明的保护范围，凡未脱离本发明技术应用精神和实物所为的等效实施或变更，均应包含于本申请的保护范围中。
Claims (2)
1.三七多糖SQP20在制备治疗肠损伤与炎性浸润的药物的应用，其特征是，所述三七多糖SQP20的分子量为4.84×105Da，单糖组成为甘露糖、鼠李糖、葡萄糖和半乳糖，摩尔比为3.78:1.89:75.81:18.52，具有Glcp-(1→、→4)-Manp-(1→、→3)-Rhap-(1→作为侧链链接在葡萄糖半乳聚糖的(1→4)-Glcp的O-6上。
2.权利要求1所述的三七多糖SQP20在制备治疗肠损伤与炎性浸润的药物的应用，其特征是，所述三七多糖SQP20由以下方法提取而得：取三七粉按固液比为1﹕10g/mL加入蒸馏水在80℃下提取3次，每次提取2h，合并3次提取液，在50℃的减压下在旋转蒸发器中浓缩到约5L，用AB-8大孔树脂脱色，用Sevag法脱蛋白，然后进行浓缩，在浓缩液中加入乙醇至乙醇浓度为20％，静置沉降，过滤获得沉淀物，即为三七多糖SQP20。
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