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Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is pathologically characterized by the deposition of β-amyloid (βA) peptides in senile plaques and neurofibrillary tangles in the brain. Flavonoids have recently been used to prevent and treat a variety of neurodegenerative diseases, but little is known about bioflavonoids. In this study, we evaluate whether a biflavonoid fraction (BF) exerts neuroprotective effects on an aged triple transgenic mouse mode of AD (3xTg-AD). Then, 21–24-month-old 3xTg AD mice were i.p. injected with 25 mg/kg of a BF from Garcinia madruno composed of morelloflavone (65%), volkensiflavone (12%), GB 2a (11%), fukugiside (6%) and amentoflavone (0.4%) every 48 h for 3 months. The BF treatment reduced βA deposition in different regions of the brain (the hippocampus, entorhinal cortex and amygdala), reduced βA1-40 and βA1-42 levels, BACE1-mediated cleavage of APP (CTFβ), tau pathology, astrogliosis and microgliosis in the brains of aged 3xTg-AD mice. Although the BF treatment weakly improved learning, animals treated with BF spent more time in the open arms of the elevated plus maze test and displayed greater risk assessment behavior than the control groups. In summary, the BF reverses histopathological hallmarks and reduces emotional disorders in the 3xTg-AD mouse model, suggesting that the biflavonoids from G. madruno represent a potential natural therapeutic option for AD if its bioavailability is improved.