Abstract: Lonicera japonica Thunb. (honeysuckle) is one of the most favorite medicinal and edible herbs. Its dried flower buds can serve as the primary medicinal components. This herb is also rich in bioactive constituents, including the phenolic acids, flavonoids, volatile oils, triterpene saponins, and iridoid glycosides. Collectively, the honeysuckle is then characterized by the hypoglycemic, antioxidant, anti-inflammatory, antiviral, and antibacterial properties. This research aims to extract the honeysuckle polyphenols and then systematically evaluate their biological activities. Firstly, a comparative analysis was conducted on five deep eutectic solvents (DES) against the conventional solvents. Specifically, 60% ethanol and deionized water were selected to determine the solvent system that maximized polyphenol recovery. Secondly, the single-factor experiment with response surface method (RSM) was employed to identify the optimal extraction parameters. Macroporous resin NKA-9 was utilized to purify the honeysuckle polyphenols after the solvent optimization. Liquid chromatography was employed to identify the components of the honeysuckle. Thirdly, the biological activities of the purified polyphenols were systematically evaluated via in vitro assays. Specifically, the hypoglycemic activity was assessed using α-glucosidase and α-amylase inhibitory assays, while the antioxidant capacity was quantified to measure the 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 the inflammatory factors (NO, iNOS, IL-6, and TNF-α). Anti-inflammatory was finally evaluated to identify the alterations in the protein levels within inflammatory pathways. The results revealed that the choline chloride-acetic acid (DES-2) was the optimal extraction solvent, thus yielding a polyphenol content of 109.93 mg/g. The variable ranges were also determined for the RSM optimization: solid-to-liquid ratio (1:30-1:50 g/mL), ultrasonic time (30-50 min), and temperature (40-60 °C). The optimal conditions were then obtained after optimization: a solid-to-liquid ratio of 1:38 g/mL, a temperature of 47 °C, and an ultrasonic time of 40 min, under which the polyphenol yield reached 121.22 mg/g. The relatively influential factors on the extraction efficiency were ranked in descending order of: ultrasonic time > solid-to-liquid ratio > temperature. Among them, the primary components of the purified honeysuckle polyphenols were extracted as the neochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C. In the concentration range of 10~50 μg/mL, the polyphenols exhibited a significant concentration-dependent inhibition on the α-glucosidase (P< 0.05), with the inhibition rates increasing from 33.53% to 76.96%. Notably, the maximum inhibition rate was approached 77.72% of by acarbose at the same concentration (50 μg/mL). At a concentration of 2 500 μg/mL, the polyphenols and acarbose exhibited the inhibition rates of 26.96% and 89.95%, respectively, against α-amylase activity. As such, the honeysuckle polyphenols shared the superior inhibitory activity on the α-glucosidase, indicating the hypoglycemic activity. In terms of the antioxidant activity, the DPPH radical scavenging rate of the polyphenols was 95.12% at a concentration of 30 μg/mL, comparable with that of vitamin C (Vc). Moreover, the ABTS radical scavenging rate of polyphenols increased from 25.67% to 99.72% in a concentration-dependent manner, thereby paralleling the Vc radical scavenging rate at a concentration of 30 μg/mL. A strong antioxidant capacity was then achieved after treatment. Polyphenols significantly inhibited the nitric oxide (NO) production at the concentrations ranging from 25 to 200 μg/mL, compared with the lipopolysaccharide (LPS)-induced positive controls (P<0.05). Furthermore, the expression of pro-inflammatory factors was significantly reduced in the LPS-induced honeysuckle polyphenol group at the cellular mRNA level, compared with the positive control group, including the inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) (P<0.05). Western blot analysis revealed that there was a gradual decrease in the intensity of phosphorylated IκB (p-IκB) and p65 (p-p65) protein bands, compared with the positive control group. Therefore, the honeysuckle polyphenols shared anti-inflammatory properties to regulate the phosphorylation of IκB and p65. These findings can be used to attenuate he activation of the nuclear factor kappa B (NF-κB) signaling pathway.