Abstract: Lonicera japonica Thunb. (honeysuckle) is a traditional medicinal and edible herb, with its dried flower buds serving as the primary medicinal part. It is rich in bioactive constituents, including phenolic acids, flavonoids, volatile oils, triterpene saponins, and iridoid glycosides. Collectively, these components endow honeysuckle with hypoglycemic, antioxidant, anti-inflammatory, antiviral, and antibacterial properties. To optimize the extraction of honeysuckle polyphenols and systematically evaluate their biological activities. Initially, this research conducted a comparative analysis of five deep eutectic solvents (DES) against conventional solvents, specifically 60% ethanol and deionized water, to determine the solvent system that maximizes polyphenol recovery. Subsequently, single-factor experiments and response surface methodology (RSM) were employed to identify the optimal extraction parameters. After optimizing the solvent, Macroporous resin NKA-9 was utilized for the purification of honeysuckle polyphenols, and liquid chromatography was employed to identify the components of honeysuckle. Next, the biological activities of the purified polyphenols were systematically evaluated through in vitro assays. Specifically, hypoglycemic activity was assessed using α-glucosidase and α-amylase inhibitory assays, while antioxidant capacity was quantified by measuring free radical scavenging rates against (1,1-diphenyl-2-trinitrophenylhydrazine) DPPH and (2,2-azinobis(3-ethylbenzothiazolino-6-sulfonic acid) diammonium salt) ABTS radicals. An LPS-induced inflammatory cell model was established to measure the cellular expression levels of inflammatory factors (NO, iNOS, IL-6, TNF-α) and to identify alterations in protein levels within inflammatory pathways for the evaluation of anti-inflammatory effects.The results revealed that choline chloride-acetic acid (DES-2) was the optimal extraction solvent, yielding a polyphenol content of 109.93 mg/g. Single-factor experiments established the following variable ranges for response surface methodology (RSM) optimization: solid-to-liquid ratio (1:30～1:50 g/mL), ultrasonic time (30～50 min), and temperature (40～60℃). RSM analysis determined the optimal conditions to be a solid-to-liquid ratio of 1:38 g/mL, a temperature of 47℃, and an ultrasonic time of 40 min, under which the polyphenol yield reached 121.22 mg/g. The relative influence of these factors on extraction efficiency, ranked from greatest to least, was as follows: ultrasonic time > solid-to-liquid ratio > temperature. Under optimal extraction conditions, the primary components of purified honeysuckle polyphenols include neochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C. In the concentration range of 10～50 μg/mL, the extracted polyphenols exhibited a significant concentration-dependent inhibitory effect on α-glucosidase (P < 0.05), with inhibition rates increasing from 33.53% to 76.96%. Notably, the maximum inhibition rate approached 77.72% of that achieved by acarbose at the same concentration (50 μg/mL). At a concentration of 2.5 mg/mL, polyphenols and acarbose exhibited inhibition rates of 26.96% and 89.95%, respectively, against α-amylase activity. The results indicated that honeysuckle polyphenols exhibited a superior inhibitory effect on α-glucosidase and demonstrated hypoglycemic activity. In terms of antioxidant activity, the DPPH radical scavenging rate of polyphenols at a concentration of 30 μg/mL was 95.12%, comparable to that of vitamin C (Vc) at the same concentration. Moreover, the ABTS radical scavenging rate of polyphenols increased from 25.67% to 99.72% in a concentration-dependent manner, paralleling the Vc radical scavenging rate at a concentration of 30 μg/mL, thereby indicating a strong antioxidant capacity. Polyphenols significantly inhibited nitric oxide (NO) production at concentrations ranging from 25 to 200 μg/mL compared to lipopolysaccharide (LPS)-induced positive controls (P<0.05). At the cellular mRNA level, the expression of pro-inflammatory factors in the LPS-induced honeysuckle polyphenol group was significantly reduced compared to the positive control group, including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) (P<0.05). Western blot analysis revealed that the intensity of phosphorylated IκB (p-IκB) and p65 (p-p65) protein bands gradually decreased in comparison to the positive control group. These findings suggest that honeysuckle polyphenols exert anti-inflammatory effects by regulating the phosphorylation of IκB and p65, thereby attenuating the activation of the nuclear factor kappa B (NF-κB) signaling pathway.