CN113730448B - 一种猴头菇抗人结肠癌肿瘤细胞（hct-8）提取物的制备方法 - Google Patents
一种猴头菇抗人结肠癌肿瘤细胞（hct-8）提取物的制备方法 Download PDFInfo
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Abstract
本发明提供一种猴头菇抗人结肠癌肿瘤细胞(HCT‑8)提取物的制备方法，采用超临界CO2萃取装置萃取得到猴头菇提取物，其制备成本相对较低，所制备的提取物对人结肠癌肿瘤细胞(HCT‑8)增殖抑制活性强，且工序简单，无溶剂残留，食用安全，既可以开发保健功能食品，也可以开发药品，因此本发明具有很好的推广价值和市场潜力。同时，本发明也提供了以开发猴头菇精深加工新方向，丰富了猴头菇高值化加工技术，为建立和完善其他食用菌多级联产的加工技术体系奠定基础。本发明普遍适用于猴头菇。
Description
技术领域
本发明提供一种猴头菇抗人结肠癌肿瘤细胞(HCT-8)提取物的制备方法，属农产品加工技术领域。
背景技术
猴头菇(Hericium erincaceus)属于担子菌亚门、担子菌纲、多孔菌目、齿菌科猴头属的大型食用菌和药用菌，俗称猴头菇、刺猬菇、山伏菌、猴头蘑、菜花菌、对脸蘑。我国是猴头的重要产地，东北各省和河南、河北、西藏、山西、四川等地都有出产。猴头营养丰富，包含丰富的蛋白质、脂肪、纤维素、多糖，还有16种氨基酸，其中8种人体必需氨基酸，是著名的药食两用菌，它质嫩味鲜，是筵席上的佳肴，人们常将其与熊掌、海参、鱼翅并列为四大名菜，并有“山珍猴头，海味燕窝”之说。猴头菌有独特的药用价值，中医认为性平，味甘，利五脏，助消化，滋补，治疗神经衰弱。研究发现，它具有抗溃疡、抗炎症、抗衰老，保肝护肝、降血糖、降血脂和降血压等疗效，开发应用前景广阔。
目前，有相关文献报道了猴头菇多糖在预防和治疗结肠癌上的应用，有文献介绍了一种成分中包括猴头菇调理大肠癌的茶，报道了猴头菌提取物对葡聚糖硫酸钠(DSS)诱导的小鼠结肠炎有改善作用，以及涉及猴头菇发酵液抑制幽门螺杆菌的研究。相关文献报道了从猴头菇子实体和菌丝体中提取物大鼠嗜铬细胞瘤PC12细胞的神经毒性有保护作用。
在猴头菇提取研究方面，有文献公开了一种猴头菇体外高效抗氧化组分及其提取方法与应用，该提取方法以猴头菇子实体为原材料，经过干燥、粉碎，过筛后乙醇溶液加热提取。相关文献介绍了制备猴头菇子实体提取物及包含该提取物的泡腾片的方法和应用，具有抗氧化，降血糖，增强免疫、抗衰老、维护胃肠道健康等功效。有文献采用复合酶法辅助热水浸提对猴头菇/ 香菇β-葡聚糖进行提取，通过正交实验对其提取工艺条件进行优化。有文献采用温和的化学顺序萃取法从猴头菇渣中提取了一种新型真菌几丁质，并且研究了脱蛋白条件(NaOH浓度、反应温度、反应时间)对甲壳素收率、纯度、分子量和乙酰化程度的影响。但所用提取工艺并非超临界CO2萃取法。
综上所述，目前猴头菇的基础研究主要集中在药理和保健功效方面，且猴头菇的功能活性有很大的开发空间，但目前仍没有关于本发明申请的方法所制备的提取物具有抗结肠癌活性的相关报道。因此，结合前期对食用菌高值化精深加工的研究基础，进行猴头菇富集抗结肠癌活性物质的研究很有必要，以开发猴头菇精深加工新方向，丰富猴头菇高值化加工技术，为建立和完善其他食用菌多级联产的加工技术体系奠定基础。
发明内容
本发明的目的是提供一种猴头菇抗人结肠癌肿瘤细胞(HCT-8)提取物的制备方法，包括以下步骤：
1)先将猴头菇干品粉碎成20～120目；
2)将上述步骤1)中粉碎后的猴头菇粉，放入超临界CO2萃取装置的萃取釜中，设定超临界CO2萃取条件参数，待萃取结束，得到猴头菇超临界CO2萃取物。其中，超临界萃取条件参数为：萃取压力 20～40Mpa，萃取温度40～50℃，分离釜I压力7-9Mpa，温度30℃-40℃，分离釜II压力3-5Mpa，温度20℃-30℃，夹带剂无水乙醇4～10mL·g-1，萃取时间0.5～2.5h；
3)将上述步骤2)中所得的猴头菇超临界CO2萃取物进行真空浓缩，待回收全部无水乙醇后，浓缩结束，得到浓缩后猴头菇提取物。其中浓缩温度为60～70℃，真空度0.5～0.6MPa。
4)将上述步骤3)中所得浓缩后猴头菇提取物，通过0.2～0.8 μm醋酸纤维素(CA)膜过滤后，收集滤液。
通过上述步骤，即制备成了一种猴头菇抗人结肠癌肿瘤细胞(HCT-8) 提取物。
本发明提供一种猴头菇抗人结肠癌肿瘤细胞(HCT-8)提取物的制备方法，是根据猴头菇的生理生长特性，结合农产品加工原料学、天然产物研究，生物化学等专业基础，在解决猴头菇多级联产体系中的关键技术环节过程中所发明的。通过使用本发明制备的提取物，对人结肠癌肿瘤细胞(HCT-8) 体外生长的抑制作用极强。研究表明，通过本发明方法所制备的猴头菇提取物，复筛的IC50值(半抑制浓度)为0.17～0.21μg/ml，对HCT-8细胞增殖抑制率达到了70％及以上，其中最佳提取条件下得到的提取物，在终浓度为 5μg/ml时，对HCT-8细胞增殖抑制率达到了71.67％，复筛的IC50值(半抑制浓度)为0.17μg/ml，相同浓度的阳性药5-氟尿嘧啶抑制率为74.06％，复筛的IC50值(半抑制浓度)为1.59μg/ml，可见萃取物的抗结肠癌肿瘤活性非常明显。
从图2和图3中可以看出，经由超临界CO2萃取后的猴头子实体粉有明显的外观和显微结构变化。食用菌菌丝体有着极为坚固的细胞壁，在 20-40MPa的压力状态下，细胞壁加压与减压时出现了明显的结构变化。至于处于超临界CO2中浸没的菌丝体细胞壁是否有通透性的变化，尚需要进一步的研究。超临界CO2萃取法有助于提高猴头子实体细胞壁的破碎程度，更利于进一步加工和人体吸收利用。将猴头菇超临界萃取物进行了分离、真空浓缩，挥干溶剂后，进行了初步的GC-MS分析检测，结果表明：猴头菇萃取物含有棕榈酸、亚油酸乙酯、亚油酸等。
本发明提供的一种猴头菇抗人结肠癌肿瘤细胞(HCT-8)提取物的制备方法，其制备成本相对较低，所制备的提取物抗人结肠癌肿瘤细胞(HCT-8) 活性强，且工序简单，无溶剂残留，食用安全，既可以开发保健功能食品，也可以开发药品，因此本发明具有很好的推广价值和市场潜力。同时，本发明也提供了以开发猴头菇精深加工新方向，丰富了猴头菇高值化加工技术，为建立和完善其他食用菌多级联产的加工技术体系奠定基础。
附图说明
图1：猴头菇萃取液(图中从左至右依次为分离釜I，II第1次取样，分离釜I，II第2次取样)。
图2：猴头子实体粉提取前后外观对比(a.提取前猴头菇子实体粉；b. 提取后猴头菇子实体粉)。
图3：猴头子实体粉提取前后显微结构对比(a.提取前猴头菇子实体粉； b.提取后猴头菇子实体粉)。
图4：猴头菇超临界CO2萃取物GC-MS的检测。
图5：猴头菇提取物对mRNA表达的影响。
图6：猴头菇提取物对小鼠体重的影响。
图7：猴头菇提取物对小鼠肿瘤体积的影响。
具体实施方式
下面通过具体实施例对本发明的加工方法进行说明，但本发明并不局限与此。所述试剂和材料和设备，如无特殊说明，均可从商业途径获得。
实施例1
一种猴头菇抗人结肠癌肿瘤细胞(HCT-8)提取物的制备方法，包括以下步骤：
1)先将猴头菇干品粉碎成20目；
2)将上述步骤1)中粉碎后的猴头菇粉，放入超临界CO2萃取装置的萃取釜中，设定超临界CO2萃取条件参数，待萃取结束，得到猴头菇超临界CO2萃取物，其中，超临界萃取条件参数为：萃取压力 40Mpa，萃取温度50℃，分离釜I压力9Mpa，温度40℃，分离釜II 压力5Mpa，温度30℃，夹带剂无水乙醇4mL·g-1，萃取时间2.5h；
3)将上述步骤2)中所得的猴头菇超临界CO2萃取物进行真空浓缩，待回收全部无水乙醇后，浓缩结束，得到浓缩后猴头菇提取物。其中浓缩温度为70℃，真空度0.50MPa；
4)将上述步骤3)中所得浓缩后猴头菇提取物，通过0.8μm 醋酸纤维素(CA)膜过滤后，收集滤液。
通过上述步骤，即制备成了一种猴头菇抗人结肠癌肿瘤细胞(HCT-8) 提取物。
实施例2.
一种猴头菇抗人结肠癌肿瘤细胞(HCT-8)提取物的制备方法，包括以下步骤：
1)先将猴头菇干品粉碎成60目；
2)将上述步骤1)中粉碎后的猴头菇粉，放入超临界CO2萃取装置的萃取釜中，设定超临界CO2萃取条件参数，待萃取结束，得到猴头菇超临界CO2萃取物，其中，超临界萃取条件参数为：萃取压力30Mpa，萃取温度45℃，分离釜I压力8Mpa，温度35℃，分离釜II压力4Mpa，温度25℃，夹带剂无水乙醇9mL·g-1，萃取时间2.0h；
3)将上述步骤2)中所得的猴头菇超临界CO2萃取物进行真空浓缩，待回收全部无水乙醇后，浓缩结束，得到浓缩后猴头菇提取物。其中浓缩温度为60℃，真空度0.6MPa；
4)将上述步骤3)中所得浓缩后猴头菇提取物，通过0.2μm 醋酸纤维素(CA)膜过滤后，收集滤液。
通过上述步骤，即制备成了一种猴头菇抗人结肠癌肿瘤细胞(HCT-8) 提取物。
实施例3.
一种猴头菇抗人结肠癌肿瘤细胞(HCT-8)提取物的制备方法，包括以下步骤：
1)先将猴头菇干品粉碎成120目；
2)将上述步骤1)中粉碎后的猴头菇粉，放入超临界CO2萃取装置的萃取釜中，设定超临界CO2萃取条件参数，待萃取结束，得到猴头菇超临界CO2萃取物，其中，超临界萃取条件参数为：萃取压力 20Mpa，萃取温度40℃，分离釜I压力7Mpa，温度30℃，分离釜II 压力3Mpa，温度20℃，夹带剂无水乙醇10mL·g-1，萃取时间0.5h；
3)将上述步骤2)中所得的猴头菇超临界CO2萃取物进行真空浓缩，待回收全部无水乙醇后，浓缩结束，得到浓缩后猴头菇提取物。其中浓缩温度为60℃，真空度0.5MPa；
4)将上述步骤3)中所得浓缩后猴头菇提取物，通过0.5μm 醋酸纤维素(CA)膜过滤后，收集滤液。
通过上述步骤，即制备成了一种猴头菇抗人结肠癌肿瘤细胞(HCT-8) 提取物。
对比例1.
将猴头菇替换为猴头菌菌丝体，其余步骤同实施例1。
对比例2.
将实施例1的步骤2)-3)替换为加入猴头菇粉10倍的去离子水加热，提取30min后冷却过滤，滤液浓缩后加入10倍无水乙醇溶解，减压浓缩。
对比例3.
将猴头菇替换为杏鲍菇，其余步骤同实施例1。
对比例4.
将猴头菇替换为双孢菇，其余步骤同实施例1。
实验一HCT-8肿瘤细胞体外生长抑制实验
选用对数生长期细胞，胰酶消化后用含10％胎牛血清RPMI1640培养液，接种在96孔培养板中，每孔细胞密度为4000个/100μl，37℃，5％CO2 培养24h。实验前换液成190μl新鲜完全培养基，实验组加入初筛浓度的样品10μl(终浓度单体化合物5μg/ml；混合物50μg/ml)，对照组则换含等体积溶剂的培养液，37℃，5％CO2培养3天，弃去培养基。轻轻加入4℃预冷的10％TCA(1640无血清培养基配制)100μl将细胞固定在培养板上。先静置5min然后再移至4℃放置1h。倒掉固定液，蒸馏水洗5次去除TCA，空气干燥(至少1小时)。每孔加入0.4％SRB溶液80μl，室温染色30min，弃染液，1％TCA(去离子水配制)洗6遍充分去除未结合的SRB，空气干燥。加入150μl 10mM非缓冲Tris碱液(pH10.5)溶解，微量振荡器上5min。酶标仪M5检测仪测定OD510nm值。如初筛结果显示样品对HCT-8细胞增殖抑制率超过50％，再对其进行复筛，并求IC50值。
肿瘤细胞生长抑制率(％)＝(OD对照-OD实验)/(OD对照-OD空白)*100％
一种猴头菇抗人结肠癌肿瘤细胞(HCT-8)提取物生物活性筛选结果见表1：
表1.HCT-8肿瘤细胞体外生长抑制实验(SRB法)结果
通过使用本发明制备的提取物，对人结肠癌肿瘤细胞(HCT-8)体外生长的抑制作用极强。研究表明，通过本发明方法所制备的猴头菇提取物，复筛的IC50值(半抑制浓度)为0.17～0.21μg/ml，对HCT-8细胞增殖抑制率达到了70％以上，其中最佳提取条件下得到的提取物，在终浓度为5μg/ml时，对HCT-8细胞增殖抑制率达到了71.67％，复筛的IC50值(半抑制浓度)为 0.17μg/ml，相同浓度的阳性药5-氟尿嘧啶抑制率为74.06％，复筛的IC50值 (半抑制浓度)为1.59μg/ml，可见萃取物的抗结肠癌肿瘤活性非常明显。通过与对比例的比较也可以发现，本发明采用超临界CO2萃取得到的猴头菇提取物相比于传统提取法，以及以其他菌菇或猴头菌菌丝体为原料提取均取得了预料不到的效果。
实验二细胞凋亡率测定实验
取对数生长期的HCT-8肿瘤细胞，调整细胞密度5*104个/ml，接种到 96孔细胞培养板中，移入培养箱培养24h使细胞贴壁，弃上清液，以不同组等量提取物处理细胞液48h后，用PBS洗涤，加入Annexin V 10μl和碘化丙啶(PI)5μl，避光反应15min，采用流式细胞仪定量检测，计算细胞凋亡率。将处理后的细胞按照试剂盒说明书提取各组细胞总RNA并合成cDNA，进行qRT-PCR分析，空白组不加入提取物处理。
表2.HCT-8肿瘤细胞凋亡率测定结果
促使细胞凋亡是发挥抗肿瘤生物活性的重要途径之一，表2肿瘤细胞凋亡率实验证明，猴头菇提取物能够显著提高HCT-8肿瘤细胞的凋亡比例。从图5的结果可以看出与空白组相比较，猴头菇提取物能够显著降低mRNA的表达水平。
实验三猴头菇提取物对小鼠移植瘤模型的影响
取一周龄小鼠随机分为四组，每组十只，每组小鼠质量均匀，取对数生长期的HCT-8肿瘤细胞，调整细胞密度5*104个/ml，于小鼠背部皮下注射 0.5mL细胞悬液，一周以后皮下出现结节，表明肿瘤模型建立成功，放置于室内，控制合适的光照和温度湿度，自由取水，空白组每天灌胃10g/kg生理盐水，氟尿嘧啶组每天按10mg/kg腹腔注射药物，实验组每天分别灌胃10g/kg 实施例1和实施例2制备的猴头菇提取物。连续5周每周称量各组小鼠体重，肿瘤体积及最终肿瘤质量。肿瘤体积＝肿瘤长径*与长径垂直的瘤体最宽径2。
从图6-7可以看出，空白组和氟尿嘧啶组小鼠体重均低于实施例组，表面猴头菇提取物可以减轻氟尿嘧啶的不良影响，实施例和氟尿嘧啶组肿瘤体积显著减小，肿瘤质量显著降低，表面猴头菇提取物能有效抑制肿瘤细胞的生长，同时减轻氟尿嘧啶药物带来的体重减轻。
Claims (1)
1.一种猴头菇抗人结肠癌肿瘤细胞HCT-8提取物的制备方法，其特征在于，包括以下步骤：
1）先将猴头菇干品粉碎成60目；
2）将上述步骤1）中粉碎后的猴头菇粉，放入超临界CO2萃取装置的萃取釜中，设定超临界CO2萃取条件参数，待萃取结束，得到猴头菇超临界CO2萃取物，其中，超临界萃取条件参数为：萃取压力30Mpa，萃取温度45℃，分离釜I压力 8Mpa，温度35℃，分离釜II 压力4Mpa，温度25℃，夹带剂无水乙醇9mL∙g-1，萃取时间2.0h；
3）将上述步骤2）中所得的猴头菇超临界CO2萃取物进行真空浓缩，待回收全部无水乙醇后，浓缩结束，得到浓缩后猴头菇提取物，其中浓缩温度为60℃，真空度0.6MPa；
4）将上述步骤3）中所得浓缩后猴头菇提取物，通过0.2μm醋酸纤维素CA膜过滤后，收集滤液；
5）通过上述步骤，即制备成了一种猴头菇抗人结肠癌肿瘤细胞HCT-8提取物。
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