CN112076233B - 盐肤木果实在制备治疗代谢综合征或糖尿病及其并发症的药物或保健品中的应用 - Google Patents
盐肤木果实在制备治疗代谢综合征或糖尿病及其并发症的药物或保健品中的应用 Download PDFInfo
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Abstract
本发明公开了食品或医药技术领域的盐肤木果实在制备治疗代谢综合征或糖尿病及其并发症的药物或保健品中的应用。本发明创造性地发现盐肤木果实或其提取物具有改善胰岛素抵抗和抑制α‑葡萄糖苷酶的作用,并经过体外化学实验和动物实验证实盐肤木果实或其提取物具有极强的α‑葡萄糖苷酶抑制活性,并能够极其显著地改善高糖高脂饮食导致的大鼠机体胰岛素抵抗状态,可有效控制血糖,用于预防、缓解或治疗代谢综合征或糖尿病及其并发症的新药物和新保健品的制备与开发。
Description
技术领域
本发明属于食品或医药技术领域,具体涉及盐肤木果实在制备治疗代谢综合征或糖尿病及其并发症的药物或保健品中的应用。
背景技术
糖尿病(diabetes mellitus,DM)是以高血糖为特征的代谢性疾病,临床表现为多饮、多尿、多食、消瘦、疲乏无力等,其引发的慢性并发症遍及全身重要器官,包括糖尿病酮症酸中毒、糖尿病高渗非酮症昏迷、糖尿病视网膜病变和糖尿病肾病等。糖尿病可以分为I型糖尿病和II型糖尿病。I型糖尿病主要是由于胰岛B细胞破坏,胰岛素绝对缺乏导致。II型糖尿病主要是以胰岛素抵抗为主伴有胰岛素相对性缺乏或胰岛素分泌受损为主伴有胰岛素抵抗。I型糖尿病多在25岁以前的青少年期起病。II型糖尿病多见于中老年人,肥胖者发病率相对较高,并且随着我国人民生活水平不断增高,高热量饮食和较少运动量的生活方式导致人群中糖尿病及其并发症的发病率明显上升。糖尿病很难根治,但患者可以通过控制血糖,延缓疾病进程。
II型糖尿病的发病基础是胰岛素抵抗,胰岛素作用的靶器官对胰岛素的敏感性下降,即正常剂量的胰岛素产生低于正常生物学效应的状态,导致人体内正常的胰岛素剂量无法正常控制血糖。此时,为了达到控制血糖的效果,机体会分泌更多的胰岛素,从而导致高胰岛素血症,长此以往,人体的胰岛功能被破坏,进而需要通过注射胰岛素来控制血糖。
目前预防和治疗糖尿病的主要方法为:(1)抑制碳水化合物在肠道中的消化水解,减缓和控制餐后血糖,这类药物以阿卡波糖为代表,是一类α-葡萄糖苷酶的抑制剂;(2)改善机体胰岛素抵抗状态,提高机体对胰岛素的敏感性,这类药物以罗格列酮为代表,是一类胰岛素增敏剂。但是这些化学药物虽然具有较好的疗效,但通常也会伴有较为严重的副作用。
因此,从天然可食用的药食两用植物中寻找和开发能有效防治糖尿病的新型天然产物或原料具有极其重要的意义。
盐肤木(Rhus chinensis Mill.)果实是漆树科植物盐肤木的果实。为我国中南和西南常见的野生阳性树,分布于东北、河北、山东、云南、湖南、陕西、甘肃等地。盐肤木果实为球形,略压扁,径约4—5mm,具有柔毛和腺毛,成熟时呈红色,果核径约3—4mm,果期10月。盐麸子早在古代就用于治疗疾病,如:《纲目》中记载,盐麸子有生津降火、化痰、润肺滋肾、消毒、治风湿、眼病等功效。盐肤木果实不仅可以作为草药使用,而且在云南多个地区,盐肤木果实还可以作为鲜果、调味品和饮料供日常食用,具有悠久的食用历史。而且在2013年,盐肤木果油已经被批准作为新食品原料,可以作为日常食用油使用。
目前,未见对盐肤木果实提取物抑制α-葡萄糖苷酶和改善胰岛素抵抗的相关研究的报道。
发明内容
本发明的目的在于提供盐肤木果实在制备治疗代谢综合征或糖尿病及其并发症的药物或保健品中的应用。
盐肤木果实或其提取物在改善胰岛素抵抗中的应用。
盐肤木果实或其提取物在制备改善胰岛素抵抗的药物或保健品中的应用。
盐肤木果实或其提取物在抑制α-葡萄糖苷酶中的应用。
盐肤木果实或其提取物在制备抑制α-葡萄糖苷酶的药物或保健品中的应用。
盐肤木果实或其提取物在制备预防、缓解或治疗代谢综合征或糖尿病及其并发症的药物或保健品中的应用。
所述糖尿病为II型糖尿病。
所述盐肤木果实提取物为水提取物、有机溶剂-水提取物、游离态多酚提取物、酯化态多酚提取物和结合态多酚提取物中的一种或两种以上。
所述盐肤木果实提取物为水提取物、有机溶剂-水提取物或游离态、结合态和酯化态多酚提取物。
所述有机溶剂-水提取物为乙醇-水提取物。
盐肤木果实的水提取物的制备方法为:盐肤木果实于沸水中熬制,离心后的上清液进行抽滤、浓缩和冻干处理,即得;
盐肤木果实的有机溶剂-水提取物的制备方法为:盐肤木果实于有机溶剂-水混合液中进行超声处理,离心后的上清液进行浓缩和冻干处理,即得;
盐肤木果实的游离态多酚提取物的制备方法为:采用甲醇-丙酮提取盐肤木果实,离心后的上清液用乙醚-乙酸乙酯萃取,将有机相层旋蒸,水相冻干,即得;
盐肤木果实的酯化态多酚提取物的制备方法为:采用甲醇-丙酮提取盐肤木果实,离心后的上清液用乙醚-乙酸乙酯萃取,水相经NaOH水解,再经乙醚-乙酸乙酯萃取,将有机相层旋蒸,水相冻干,即得;
盐肤木果实的结合态多酚提取物的制备方法为:采用甲醇-丙酮提取盐肤木果实,离心后的下层固体沉淀用NaOH水解,取水相乙醚-乙酸乙酯萃取,将有机相层旋蒸,水相冻干,即得。
本发明技术方案,具有如下优点:
1、本发明创造性地发现盐肤木果实或其提取物具有改善胰岛素抵抗和抑制α-葡萄糖苷酶的作用,并经过体外化学实验和动物实验证实盐肤木果实或其提取物具有极强的α-葡萄糖苷酶抑制活性,并能够极其显著地改善高糖高脂饮食导致的大鼠机体胰岛素抵抗状态,可有效控制血糖,用于预防、缓解或治疗代谢综合征或糖尿病及其并发症的新药物和新保健品的制备与开发。
2、盐肤木植物可以适应各种恶劣的自然环境,在我国的分布非常的广泛,其果实价格非常的低廉;在云南等地盐肤木果实经常被用作调味品和饮品的制作原料,是一种传统药食两用的果实,而且盐肤木果油已于2013年被批准为新食品原料,其安全性高。
盐肤木果实或其提取物用于制备预防、缓解或治疗代谢综合征或糖尿病及其并发症的新药物和新保健品,其具有来源广泛、安全性高、成本低廉、提取工艺简单等优点。
3、盐肤木果实中富含酚类物质,植物酚类物质具有抗氧化、延缓衰老和保护细胞等功效,因此,用于预防、缓解或治疗代谢综合征或糖尿病及其并发症的药物和保健品,还具有其他的健康功效。
附图说明
图1是盐肤木果实不同提取物对α-葡萄糖苷酶活性的抑制率;
图2是高糖高脂饮食导致的大鼠胰岛素抵抗症及盐肤木果实不同提取物干预后胰岛中胰岛素的免疫组化切片图(×400)及胰岛素的定量柱状图;
图3是高糖高脂饮食导致的大鼠胰岛素抵抗症及盐肤木果实不同提取物干预后的肝糖原免疫组化切片图(×200)及肝糖原的定量柱状图;
图4是高糖高脂饮食导致的大鼠胰岛素抵抗症及盐肤木果实不同提取物干预后的GLUT4免疫组化切片图(×400)及GLUT4的定量柱状图;
其中K为空白对照组;M为模型组;AL为乙醇-水提取物低剂量组;AH为乙醇-水提取物高剂量组;WL为水提取物低剂量组;WH为水提取物高剂量组。
具体实施方式
提供下述实施例是为了更好地进一步理解本发明,并不局限于所述最佳实施方式,不对本发明的内容和保护范围构成限制,任何人在本发明的启示下或是将本发明与其他现有技术的特征进行组合而得出的任何与本发明相同或相近似的产品,均落在本发明的保护范围之内。
实施例中未注明具体实验步骤或条件者,按照本领域内的文献所描述的常规实验步骤的操作或条件即可进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规试剂产品。
实施例1
通过体外生物化学实验方法,研究盐肤木果实不同提取物对α-葡萄糖苷酶的抑制能力,进而评价盐肤木果实对糖尿病的餐后血糖控制和治疗作用。
(1)盐肤木果实不同提取物的制备
水提取物:采用模拟药典中植物的煎熬特性,盐肤木果实与水的料液比为1:20,在沸水中熬制1小时;煮沸以后趁热4000r/min离心,上清液进行抽滤、浓缩、冻干,即得。
乙醇-水提取物:盐肤木果实与水的料液比为1:5,采用质量浓度80%的乙醇溶液进行超声提取,提取温度为25℃,提取时间为30min,滤渣复提一次,4000r/min离心,上清液浓缩、冻干,即得。
游离态多酚:采用质量浓度70%的甲醇-丙酮(v/v=1:1)提取盐肤木果实,离心后,取上清,用1:1的乙醚-乙酸乙酯萃取,将有机相层旋蒸,水相冻干,即得。
酯化态多酚:采用质量浓度70%的甲醇-丙酮(v/v=1:1)提取盐肤木果实,离心后,取上清,用1:1的乙醚-乙酸乙酯萃取,水相经4M的NaOH水解4小时,将pH调到2后再经1:1的乙醚-乙酸乙酯萃取,将有机相层旋蒸,水相冻干,即得。
结合态多酚:采取质量浓度70%的甲醇和质量浓度70%的丙酮(v/v=1:1)提取盐肤木果实,离心后下层固体沉淀用4M的NaOH水解4小时,将pH调到2后取水相,用1:1的乙醚-乙酸乙酯萃取,将有机相层旋蒸,水相冻干,即得。
(2)α-葡萄糖苷酶抑制实验
20μL不同浓度的盐肤木果实提取物与30μL 5.84×10-7M的α-葡萄糖苷酶混合,对照组中不含抑制剂,只含有α-葡萄糖苷酶。用PBS补齐到相同体积,37℃下孵育10min后,加入30μL 5mM的pNPG,在37℃下再孵育10min,于405nm处,每隔30秒检测反应物的吸光值变化。以时间为横坐标,吸光值为纵坐标,绘制出线性回归方程得出斜率。
α-葡萄糖苷酶抑制率通过以下公式计算:
抑制率(%)=1-(R/R0)×100%
其中,R为添加了盐肤木提取物组的斜率,
R0为对照组斜率。
盐肤木果实不同提取物对α-葡萄糖苷酶活性的抑制率如图1和表A所示。
表A
盐肤木果实不同提取物对α-葡萄糖苷酶的抑制活性如表1所示。
表1盐肤木果实不同提取物对α-葡萄糖苷酶的抑制活性
注:同一列不同字母代表具有显著性差异(p<0.05)
由表1可知,盐肤木果实不同提取物对α-葡萄糖苷酶都具有较好的抑制效果,其中游离多酚提取物以及乙醇-水提取物抑制效果尤其突出。盐肤木果实及其提取物是优良的α-葡萄糖苷酶抑制剂,能够通过强烈抑制α-葡萄糖苷酶控制餐后血糖,从而有效防治糖尿病。
实施例2
应用高糖高脂饲料诱导的方法建立大鼠胰岛素抵抗的模型,通过动物实验,观察盐肤木果实的乙醇-水提取物和水提物对大鼠胰岛素抵抗的改善作用。
(1)实验材料
SD雄性大鼠,4-8周龄,SPF级,体重在200g左右,偏差5%,购自辽宁长生生物技术股份有限公司,大鼠的编号为大鼠的编号为SCKK(辽)2015-0002。
高糖高脂饲料,购自进多丰生物有限公司。
试剂盒,购自赛默飞世尔科技公司。
(2)盐肤木果实提取物的制备
水提取物:采用模拟药典中植物的煎熬特性,盐肤木果实与水的料液比为1:20,在沸水中熬制1小时;煮沸以后趁热4000r/min离心,上清液进行抽滤、浓缩、冻干,备用。
乙醇-水提取物:盐肤木果实与水的料液比为1:5,采用质量浓度80%的乙醇溶液进行超声提取,提取温度为25℃,提取时间为30min,滤渣复提一次,4000r/min离心,上清液浓缩、冻干,备用。
(3)改善高糖高脂饮食诱导的胰岛素抵抗的实验
将36只SD雄性大鼠分成6组(每组6只),分组及编号如下:
Group K:对照组(以蒸馏水灌胃,K)、Group M:模型组(蒸馏水灌胃,M)、Group AL:乙醇-水提取物低剂量组200mg/kg(乙醇-水提取物灌胃,AL)、Group AH:乙醇-水提取物高剂量组600mg/kg(乙醇-水提取物灌胃,AH)、Group WL:水提取物低剂量组200mg/kg(水提取物灌胃,WL)、Group WH:水提取物高剂量组600mg/kg(水提取物灌胃,WH)。
第一次给药前所有大鼠禁食不禁水12h,并且记录各个大鼠的体重。按照标准的大鼠饲养环境饲养。
胰岛素抵抗是糖尿病发病的主要原因,在机体出现胰岛素抵抗后,胰岛素的靶器官对胰岛素的敏感性较低,为了有效地控制血糖,机体需要增加胰岛素的分泌量,从而会导致血液中胰岛素含量升高,产生高胰岛素血症。长期的高糖高脂饮食是诱发机体胰岛素抵抗的重要原因之一。造模的方法为:每天早上9点以5mL/kg的灌胃体积,灌入对应剂量的盐肤木果实提取物或蒸馏水;在灌胃以后,各个组给予高糖高脂饲料的喂养;在饲养90天以后,对所有的大鼠禁食12h,使用10%的水合氯醛麻醉大鼠,取血液制成血浆待测胰岛素浓度;另取胰脏和肝脏浸没在10%的福尔马林溶液中,待后续进行切片和免疫组化染色。
胰岛中胰岛素和肝脏中GLUT4的免疫组化染色:肝脏和胰脏组织浸泡在组织固定液中,用低浓度到高浓度酒精作脱水剂,逐渐脱去组织块中的水份。将脱水的组织进行石蜡包埋、切片。将切片使用二甲苯进行脱蜡处理,随后使用5%牛血清白蛋白封闭10-30分钟。使用对应的一抗抗体进行有抗体的孵育。用PBS清洗后进行二抗的孵育(二抗是HRP偶联的IgG聚合物多克隆抗体)然后用水冲洗,最后用倒置显微镜观察(Olympus IX83,Japan)。
肝脏中肝糖原的PAS染色:肝脏组织浸泡在组织固定液中,用低浓度到高浓度酒精作脱水剂,逐渐脱去组织块中的水份。将脱水的组织进行石蜡包埋、切片。然后石蜡切片脱蜡至水,蒸馏水洗,70%酒精洗。随后浸入过碘酸酒清夜10min。然后使用自来水冲洗10min,加入PAS染液10min。染色完成后,流水冲洗5min。再用苏木精(Hematoxylin,H)&伊红(Eosin,E)染色2-5min,再用无水乙醇分色与脱水,二甲苯透明,风干后中性树胶封片,在显微镜下观察(Olympus IX83,Japan)。
染色后胰岛素、糖原和GLUT4采用ImagePro plus 6.0软件进行定量。首先校准光密度,并设置感兴趣的区域(蓝,0-30;饱和,0-255;强度,0-255)。随后使用软件测量密度和面积,计算平均密度(密度平均值=密度和/面积和)。以“平均密度”对胰岛素、糖原和GLUT4进行定量。
(4)结果
a.盐麸木果实提取物对大鼠血液中胰岛素含量以及胰岛素分泌的影响
盐肤木果实提取物对大鼠血液中胰岛素含量的影响如表2所示。
表2盐麸木果实提取物对大鼠血浆中胰岛素含量的影响
注:同一列不同字母代表具有显著性差异(p<0.05)
由表2可知,与空白对照组相比,模型组大鼠血液中胰岛素含量显著增加(p<0.05),增加了约54%,说明模型组大鼠的机体出现了胰岛素抵抗症状,机体胰岛素分泌增加。而与模型组相比,盐肤木果实不同剂量的水提取物和乙醇-水提取物均可以一定程度地降低大鼠血液中的胰岛素含量,说明不同提取物能够一定程度上改善高糖高脂引起的机体胰岛素抵抗状态,尤其是高剂量的乙醇-水提取物,其可以非常显著地降低大鼠血液中胰岛素的含量(p<0.05),降低约23%。
盐肤木果实不同提取物干预后胰岛中胰岛素的免疫组化染色及胰岛素的定量结果如图2和表3所示。与空白组相比,M组大鼠胰岛β细胞周围的胰岛素分泌量明显增多,增加了约90%,而经盐肤木果实提取物处理的各组,大鼠胰岛β细胞周围的胰岛素分泌量均有一定程度的减少,尤其是AH组的减少最为明显,减少了近40%,说明盐肤木果实改善了机体胰岛素抵抗状态,从而机体可以减少胰岛素的分泌。由此可以证实盐肤木果实可以有效改善长期高糖高脂饮食引发的机体胰岛素抵抗,从而可以有效预防和改善II型糖尿病。
表3
组别 | 胰岛素平均密度 |
K | 74.86±8.00 |
M | 144.53±10.1* |
AL | 121.64±6.03* |
AH | 91.18±9.57# |
WL | 114.34±11.67*# |
WH | 100.14±7.58*# |
b.盐肤木果实提取物对糖尿病大鼠肝糖原含量的影响
在肝脏组织中,肝糖原水平的降低是胰岛素抵抗的另一个特征。葡萄糖分子以糖原聚合物的形式储存于肝脏,当机体需要氧化产能时,这些糖原便可以分解成葡萄糖,转化为能量。当机体产生胰岛素抵抗后,身体组织吸收血糖能力下降,造成机体缺少葡萄糖的假象,因此,肝脏中的肝糖原会不断分解为葡萄糖以供机体组织能量需求,从而导致肝糖原含量的减少,同时肝脏本身胰岛素抵抗后对血糖的吸收也降低,也是造成肝糖原合成减少的另一原因。
盐肤木果实不同提取物干预后的肝糖原免疫组化染色及肝糖原的定量结果如图3和表4所示。从肝糖原PAS染色结果可以看出:M组中,肝糖原含量明显低于K组(p<0.05),下降近70%。然而,与模型组相比,所有盐肤木果实提取物组的肝糖原都有一定程度的增多,尤其是AH组,增加尤为明显,增加了近150%,几乎与正常对照组相当。这说明,盐肤木果实能有效地改善机体胰岛素抵抗状态,增加肝糖原含量。
表4
组别 | 糖原平均密度 |
K | 549.08±25.23 |
M | 181.59±27.39* |
AL | 387.31±23.82*# |
AH | 465.18±22.85*# |
WL | 233.1±26.55* |
WH | 353.72±21.46*# |
c.盐肤木果实提取物对糖尿病大鼠肝脏中葡萄糖转运蛋白(GLUT4)的影响
GLUT4是葡萄糖摄取的重要蛋白,其表达量的降低是导致机体胰岛素抵抗的主要原因之一。
盐肤木果实不同提取物干预后的GLUT4免疫组化染色及GLUT4的定量结果如图4和表5所示。从肝脏GLUT4的免疫组化结果可以看出,与空白对照组相比,M组的GLUT4蛋白表达明显低于K组,下降近60%。而相较于模型组,几乎所有盐肤木果实提取物干预组大鼠肝脏中GLUT4都有一定程度的增多,尤其是AH组,增加最为明显,上升了约95%,几乎与空白对照组相当。这说明,盐肤木果实提取物能有效增加GLUT4的含量,从而改善高糖高脂所引起的机体胰岛素抵抗。
表5
组别 | GLUT4平均密度 |
K | 92.12±2.53 |
M | 40.04±4.73* |
AL | 62.79±3.76*# |
AH | 77.31±4.82*# |
WL | 43.08±2.91* |
WH | 53.66±3.4*# |
综上所述:可以确定盐肤木果实可以有效的抑制α-葡萄糖苷酶活性,减缓摄入的碳水化合物的消化,减缓葡萄糖的吸收,从而改善控制糖尿病餐后血糖。另外,盐肤木果实也可以改善长期高糖高脂饮食引起的机体胰岛素抵抗,预防和治疗II型糖尿病。从而确定盐肤木果实可以有效地防治二型糖尿病及相关并发症。
显然,上述实施例仅仅是为清楚地说明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (3)
1.盐肤木果实提取物在制备具有降血糖功效的保健品中的应用;所述盐肤木果实提取物为游离态多酚提取物,盐肤木果实的游离态多酚提取物的制备方法为:采用质量浓度70%的甲醇-丙酮v/v=1:1提取盐肤木果实,离心后,取上清,用1:1的乙醚-乙酸乙酯萃取,将有机相层旋蒸,水相冻干,即得。
2.盐肤木果实提取物在制备缓解或治疗糖尿病的药物中的应用;所述盐肤木果实提取物为游离态多酚提取物,盐肤木果实的游离态多酚提取物的制备方法为:采用质量浓度70%的甲醇-丙酮v/v=1:1提取盐肤木果实,离心后,取上清,用1:1的乙醚-乙酸乙酯萃取,将有机相层旋蒸,水相冻干,即得。
3.根据权利要求2所述的应用,其特征在于,所述糖尿病为II型糖尿病。
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