Protective Effect Of Trigonella Foenum-graecum Flavonoids On Chronic Restraint-induced Neurological Stress

The body usually responds constantly under physiological conditions to maintain physiological functions within a certain range,but long-term stress affects the Central Nervous System（CNS）and the Hypothalamic-Pituitary-Adrenal axis（HPA）.Injury of neurons leads to mental confusion and symptoms such as depression or cognitive impairment.Therefore,the search for active substances with neuroprotective effects and safety and efficacy has traditionally been a research hotspot.In this paper,the anti-stress functional components were screened from seven natural plants including Condonopsis pilosula,Trigonella foenum-graecum,Gynostemma pentaphyllum,Polygala tenuifolia,Astragalus membranaceus,Ginkgo biloba leaves and Radix paeoniae alba by the method of bioactivity-guided isolation.The protective effect and mechanism of functional components on neurological stress were studied by establishing PC12 cell injury model and mouse chronic restraint stress model.The inhibitory model of acetylcholinesterase and monoamine oxidase-A in vitro was established.Column chromatography was used to isolate and purify the extract.The fenugreek seed flavonoids（FSF）from seven natural extracts with the highest inhibitory rate(IC₅₀=25.80μg/mL and 190.59μg/mL).LC-MS/MS analysis showed that the composition and relative percentage of the main active substances in FSF were as follows:Kaempferol 3-（p-coumaryl）glucoside:Quercetin 4′-O-β-d-glucopyranoside:Apigenin4′,7-O-diglucoside:Schaftoside:Isoschaftoside:Apigenin 8-C-α-D-glucopyranoside=5.58:16.54:21.98:30.94:19.2:5.76.The corticosterone-induced PC12 cell injury model was established to evaluate the protective effect of FSF.Compared with CORT group,FSF can increase cell viability（86.8±6.4%,P<0.01）,reduce LDH release rate（44.7±4.7%,P<0.01）,decrease the number of apoptosis,lower the activity of AChE（15.40±1.58 U/10⁴ cell,P<0.01）and MAO-A（115.4±7.6%,P<0.01）,and finally prove that FSF has the neuroprotective effect on damaged PC12 cells.The mice model of chronic restraint stress was established.The neuroprotective effects and mechanisms of FSF were studied by behavioral tests and biochemical indicators.The results showed that FSF can effectively restore the depression-like behavior caused by chronic restraint stress by reducing the immobility time of mice forced swimming test and tail suspension experiment（P<0.05,P<0.01）and increasing the preference of sugar water（P<0.01）.FSF significantly decreased serum corticosterone levels induced by chronic restraint stress（P<0.01）,increased prefrontal cortex neurotransmitters（ACh,NE,5-HT and DA）,hippocampus neurotransmitters（ACh,NE,5-HT and DA）and striatum neurotransmitter（NE）levels（P<0.05,P<0.01）.FSF also showed significant inhibitory effects on AChE and MAO-A activities in prefrontal cortex and hippocampus（P<0.01）.FSF significantly down-regulated the expression levels of KLF11,SIRT1 and MAO-A in prefrontal cortex and hippocampus,inhibited the expression and activity of MAO-A through KLF11/SIRT1-MAO-A pathway,and increased the level of monoamine neurotransmitters,thus protecting against chronic restraint-induced nerve stress.This study reveals the protective effect and mechanism of FSF on nerve stress injury,and has a certain reference significance for the development of new anti-stress natural products.