Ponnusamy Sudha, Haldar Saikat, Mulani Fayaj, Zinjarde Smita, Thulasiram Hirekodathakallu, RaviKumar Ameeta
Institute of Bioinformatics and Biotechnology, Savitribai Phule Pune University, Pune 411 007, Maharashtra, India.
Chemistry-Biology Unit, Division of Organic Chemistry, CSIR-National Chemical Laboratory, Pune 411 008, Maharashtra, India.
PLoS One. 2015 Oct 15;10(10):e0140113. doi: 10.1371/journal.pone.0140113. eCollection 2015.
Human pancreatic α-amylase (HPA) inhibitors offer an effective strategy to lower postprandial hyperglycemia via control of starch breakdown. Limonoids from Azadirachta indica known for their therapeutic potential were screened for pancreatic α-amylase inhibition, a known anti-diabetic target. Studies were carried out to reveal their mode of action so as to justify their hypoglycemic potential. Of the nine limonoids isolated/semi-synthesized from A.indica and screened for α-amylase inhibition, azadiradione and exhibited potential inhibition with an IC50 value of 74.17 and 68.38 μM, respectively against HPA under in vitro conditions. Further screening on AR42J α-amylase secretory cell line for cytotoxicity and bioactivity revealed that azadiradione and gedunin exhibited cytotoxicity with IC50 of 11.1 and 13.4μM. Maximal secreted α-amylase inhibition of 41.8% and 53.4% was seen at 3.5 and 3.3μM, respectively. Michaelis-Menten kinetics suggested a mixed mode of inhibition with maltopentaose (Ki 42.2, 18.6 μM) and starch (Ki' 75.8, 37.4 μM) as substrate with a stiochiometry of 1:1 for both azadiradione and gedunin, respectively. The molecular docking simulation indicated plausible π-alkyl and alkyl-alkyl interactions between the aromatic amino acids and inhibitors. Fluorescence and CD confirmed the involvement of tryptophan and tyrosine in ligand binding to HPA. Thermodynamic parameters suggested that binding is enthalpically and entropically driven with ΔG° of -21.25 kJ mol-1 and -21.16 kJ mol-1 for azadiradione and gedunin, respectively. Thus, the limonoids azadiradione and gedunin could bind and inactivate HPA (anti-diabetic target) and may prove to be lead drug candidates to reduce/control post-prandial hyperglycemia.
人胰腺α-淀粉酶（HPA）抑制剂提供了一种通过控制淀粉分解来降低餐后高血糖的有效策略。对印楝中具有治疗潜力的柠檬苦素类化合物进行了胰腺α-淀粉酶抑制作用筛选，胰腺α-淀粉酶是一个已知的抗糖尿病靶点。开展了相关研究以揭示其作用模式，从而证明其降血糖潜力。从印楝中分离/半合成并筛选α-淀粉酶抑制作用的9种柠檬苦素类化合物中，印楝二酮和格杜宁在体外条件下对HPA表现出潜在抑制作用，IC50值分别为74.17和68.38μM。在AR42Jα-淀粉酶分泌细胞系上进一步进行细胞毒性和生物活性筛选发现，印楝二酮和格杜宁具有细胞毒性，IC50分别为11.1和13.4μM。在3.5和3.3μM时，最大分泌α-淀粉酶抑制率分别为41.8%和53.4%。米氏动力学表明，以麦芽五糖（Ki分别为42.2、18.6μM）和淀粉（Ki'分别为75.8、37.4μM）作为底物时，印楝二酮和格杜宁均以1:1的化学计量比呈现混合抑制模式。分子对接模拟表明，芳香族氨基酸与抑制剂之间存在可能的π-烷基和烷基-烷基相互作用。荧光和圆二色光谱证实色氨酸和酪氨酸参与配体与HPA的结合。热力学参数表明，结合是由焓和熵驱动的，印楝二酮和格杜宁的ΔG°分别为-21.25 kJ mol-1和-21.16 kJ mol-1。因此，柠檬苦素类化合物印楝二酮和格杜宁可结合并使HPA（抗糖尿病靶点）失活，可能成为降低/控制餐后高血糖的潜在候选先导药物。
