摘要
Objective To evaluate the in vitro anti-diabetic effects of Bryonia dioica roots extracts,in-cluding water-acetone extracts and their ethyl acetate and butanol fractions,and chloroform-methanol extracts.Methods The total phenolic,flavonoid,flavonol,and saponin contents in the Bryonia dioica root extracts(chloroform-methanol extracts,water-acetone extracts and their ethyl acetate and butanol fractions)were determined using colorimetric methods with Folin-Ciocalteu,aluminum trichloride,and vanillin reagents,respectively.The in vitro anti-diabetic activity was evaluated by measuring the half-maximal inhibitory concentration(IC_(50))values of these root extracts againstα-amylase andα-glucosidase activities,evaluating their effects onα-amy-lase kinetics,quantifying the inhibition of bovine serum albumin(BSA)glycation using fluo-rometry to assess advanced glycation end products(AGE)production,and determining glu-cose uptake by isolated rat hemidiaphragm.Additionally,molecular docking analysis was conducted to investigate the binding affinity and interaction types between Bryonia dioica lig-ands(cucurbitacin B,bryogénin,vitexin,and isovitexin)and target enzymes,and a phyto-chemical-targets interaction network was constructed.Results Forα-amylase inhibition,ethyl acetate fraction demonstrated the most potent activi-ty(IC_(50)=145.95μg/mL),followed by chloroform-methanol extract(IC_(50)=300.86μg/mL).Water-acetone root extracts and their ethyl acetate and butanol fractions inhibited theα-glucosidase activity with IC50 values ranging from 562.88 to 583.90μg/mL.Both ethyl acetate and butanol fractions strongly inhibited non-enzymatic BSA glycation(IC_(50)=318.26 and 323.12μg/mL,respectively).The incubation of isolated rat hemidiaphragms with the ethyl acetate fraction(5 mg/mL)significantly increased glucose uptake(35.16%;P<0.0001),exceeding the effects of insulin(29.27%),chloroform-methanol extract(24.07%),and catechin(15.27%).Molecular docking revealed that cucurbitacin B exhibited the strongest docking scores againstα-amylase(-16.4 kcal/mol),andα-glucosidase(-14.2 kcal/mol).Compared with other ligands,isovitexin formed the maximum number of hydrogen bonds with theα-amylase active site residues(Asp300,Asp197,and Glu233),α-glucosidase residues(Ser13,Arg44,Met86,Gly10,Asp39,and Tyr131)and other residues(Arg195,Trp59,His299,and Tyr62).Network analysis identified 36 overlapping targets between Bryonia dioica phyto-chemicals and type 2 diabetes mellitus-associated genes,with cucurbitacins and polyphenols interacting withα-amylase,α-glucosidase,and Glut4 translocation pathway targets.Conclusion Bryonia dioica root extracts demonstrated promising in vitro anti-diabetic activi-ty through multiple mechanisms,including the inhibitory effect on digestive enzymes,pro-tein antiglycation potential,and enhancement of glucose uptake,suggesting their potential as a source for anti-diabetic drugs development.
目的评价泻根根部水-丙酮提取物及其乙酸乙酯组分和丁醇组分,以及氯仿-甲醇提取物的体外抗糖尿病作用。方法分别用福-乔试剂、三氯化铝和香草醛试剂采用比色法测定泻根提取物(氯仿-甲醇提取物、水-丙酮提取物及其乙酸乙酯和丁醇组分)中的总酚、黄酮、黄酮醇和皂苷含量。通过测定这些提取物对α-淀粉酶和α-葡萄糖苷酶活性的半抑制浓度(IC_(50))值,评价其对α-淀粉酶动力学的影响,评价其体外抗糖尿病活性。采用荧光测定法定量测定对牛血清白蛋白(BSA)糖基化的抑制作用,以评估晚期糖基化终末产物的生成,并通过离体大鼠半隔膜测定葡萄糖摄取。此外,进行了分子对接分析,以研究泻根配体(葫芦素B、泻根苦素、牡荆素和异牡荆素)与α-淀粉酶和α-葡萄糖苷酶之间的结合亲和力和相互作用类型。同时构建了植物化学-靶标相互作用网络。结果对于α-淀粉酶抑制作用,乙酸乙酯部分表现出最强的活性(IC_(50)=145.95μg/mL),其次是氯仿-甲醇提取物(IC_(50)=300.86μg/mL)。水-丙酮根提取物及其组分对α-葡萄糖苷酶活性有抑制作用,IC_(50)值为562.88–583.90μg/mL。乙酸乙酯和丁醇组分强烈抑制非酶BSA糖基化,IC_(50)分别为318.26和323.12μg/mL。离体大鼠膈肌与乙酸乙酯组分(5 mg/mL)孵育显著增加葡萄糖摄取(35.16%),超过胰岛素(0.4 IU/mL;29.27%)、氯仿-甲醇提取物(24.07%)和儿茶素(15.27%)的作用。葫芦素B与α-淀粉酶(–16.4 kcal/mol)和α-葡萄糖苷酶(–14.2 kcal/mol)的对接评分最强。与其他配体相比,异牡荆素与α-淀粉酶活性位点残基(Asp300、Asp197和Glu233)和α-葡萄糖苷酶残基(Ser13、Arg44、Met86、Gly10、Asp39和Tyr131)以及其他残基(Arg195、Trp59、His299和Tyr62)形成最大数量的氢键。网络分析发现泻根植物化学成分与2型糖尿病相关基因之间存在36个重叠靶点,其中葫芦素类和多酚类化合物与α-淀粉酶、α-葡萄糖苷酶以及Glut4转位通路靶点发生相互作用。结论体外研究表明,泻根提取物具有较好抗糖尿病活性。对消化酶的抑制作用、蛋白抗糖基化潜能和葡萄糖摄取的增强提示了泻根提取物可能通过调节高血糖及其并发症的机制。该种属可能成为管理2型糖尿病的新型抗糖尿病药物的潜在来源。
作者简介
Corresponding author:Nabila Benariba,E-mail:nabila.benariba@univ-tlemcen.dz.
