Chemical composition and biological activity of ethanol gradient extracts from Turpinia arguta leaves

<bold>Objective</bold> Ethanol solutions of different concentrations were used to extract the leaves of <italic>Turpinia arguta</italic>，followed by the determination of active ingredient contents in the extracts，the evaluation of their antioxidant and anti-inflammatory activities，and the comprehensive screening of the optimal ethanol concentration with high extraction efficiency and favorable biological activities.This study is intended to provide a reference for the optimization of the crude extraction process of <italic>Turpinia arguta</italic> medicinal materials<bold>.</bold> <bold>Method</bold> The extraction was performed using water extraction-alcohol precipitation and gradient ethanol solutions（10%，30%，50%，70%，and 90%）.The contents of total polyphenols and total flavonoids were determined by ultraviolet spectrophotometry，while the concentrations of specnuezhenide，rhofolin，and ursolic acid were quantified using high-performance liquid chromatography（HPLC）.Antioxidant activities were assessed through DPPH and ABTS radical scavenging assays，as well as the ferric reducing antioxidant power（FRAP）test.Anti-inflammatory activity was evaluated by measuring the inhibitory effects of extracts on nitric oxide（NO）production in LPS-induced RAW 264.7 macrophages. <bold>Result</bold> Ethanol concentration significantly influenced the extraction efficiency of bioactive constituents.The 70% ethanol group demonstrated superior comprehensive extraction performance，yielding the highest contents of specnuezhenide，rhoifolin，ursolic acid，and total phenolics compared to other concentrations.Extracts using 90% ethanol ranked second，while the 10% ethanol group showed the lowest yields.In terms of total flavonoid content，the 90% ethanol extract exhibited the highest level among all groups；however，no significant difference（<italic>P</italic>&gt;0.05）was observed between the 90% and 70% ethanol groups.Considering solvent cost-benefit analysis，70% ethanol likely represents the optimal equilibrium point for process optimization.Antioxidant activity assays revealed significant positive correlations（<italic>P</italic>&lt;0.05）between total phenolic/flavonoid contents and antioxidant/anti-inflammatory indices.The 70% ethanol extract displayed optimal antioxidant capacity across all tests：DPPH radical scavenging rate（62.7%），ABTS radical scavenging rate（70%），and FRAP value（0.29 μmol Trolox/mL）.For anti-inflammatory activity，at a concentration of 0.075 mg/mL，both 70% and 90% ethanol extracts demonstrated substantial inhibition of NO production，with inhibition rates of 74.24% and 76.89%，respectively. <bold>Conclusion</bold> 70% ethanol represents an optimal solvent concentration for extracting active constituents from <italic>Turpinia arguta</italic> leaves.Extracts obtained using this concentration effectively enriched key bioactive compounds，including ligustroflavone，rhoifolin，ursolic acid，total phenolics，and total flavonoids（with flavonoid content comparable to 90% ethanol extracts）.Furthermore，they exhibited superior comprehensive bioactivity，demonstrating the highest antioxidant capacity（as evidenced by DPPH and ABTS radical scavenging rates，and FRAP values） and significant anti-inflammatory activity（NO inhibition rate of 74.24%）.These results indicate that 70% ethanol achieves an optimal balance between bioactive compound yield，functional activity，and economic feasibility.The high extraction efficiency likely stems from the favorable solvation properties of this medium-polarity solvent.Specifically，70% ethanol exhibits strong dissolving capacity for diverse target compounds spanning medium-polarity（e.g.，medium-polarity flavonoid glycosides），medium-to-low polarity（e.g.，triterpenoids such as ursolic acid），and polar polyphenols.This broad-spectrum solvation capability overcomes the limitations of single-polarity solvents，enabling simultaneous and efficient extraction of bioactive compounds with diverse polarity ranges from plant materials.From the perspective of industrialization，70% ethanol demonstrates distinctive advantages.Compared with high-concentration ethanol，it can reduce the solvent cost by 20%-25%.Multi-batch repeated experiments show that the relative standard deviation（RSD）of the process is less than 3%，indicating good process stability.After vacuum recovery，the residual ethanol is non-toxic and harmless，with favorable safety performance This study provides valuable data to support the standardization of the primary extraction process，quality control，and subsequent formulation development of <italic>Turpinia arguta</italic> crude drugs.Future research could further explore the synergistic interactions among the compounds in the extract and verify its activity using in vivo models.For instance，it can analyze the synergistic effect between ligustroflavone and ursolic acid via the isobologram method，establish inflammatory models such as carrageenan-induced rat paw edema for in vivo validation，develop novel drug delivery systems（e.g.，nanoemulsions）to enhance bioavailability，and construct an HPLC fingerprint with ligustrin and ursolic acid as dual marker components.From the perspective of industrial application，it is recommended to adopt a multi-stage extraction process using 70% ethanol as the primary solvent to maximize resource utilization efficiency and the comprehensive recovery of active ingredients.