Isolation On Chemical Constituents From Podocarpus Macrophllus And Their Tyrosinase Inhibition Activity

Totarol is a typical diterpene of totarane type.It was first extracted from the heartwood of Podocarpus totara by supercritical CO₂ technology.It has shown high biological activity,human body safety and compatibility of product formula,and has been included in the category of cosmeceutical products in Europe and America.In this paper,the polar parts of ethyl acetate and chloroform of P.macrophyllus,which is rich in resources,are systematically separated as raw materials.In addition,the antioxidant and tyrosinase inhibitory activities of P.macrophyllus were preliminarily explored,aiming at exploring the natural active components in P.macrophyllus and looking for compounds with similar structure and activity to toratol as substitute resources.At the same time,it also provides important scientific basis for the application of P.macrophyllus in whitening and anti-aging cosmetics.The content of total diterpene in the extract of P.macrophyllus was determined by UV spectrophotometry with totarol as the control.The linear equations were:A=0.0074C+0.0023,R²=0.9998(concentration C/?mg/mL?,absorbance/?A?.Ultrasonic assisted extraction was used to optimize the extraction process of total diterpenoids from P.macrophyllus.On the basis of single factor test and response surface methodology?Box-Behnken?design,the optimal extraction process of total diterpene from P.macrophyllus was obtained as follows:methanol concentration 80.00%,liquid-solid ratio26.00:1?mL/g?;The extraction temperature was 63.00?,the extraction time was 20 min,and the extraction time was 3 times.The total diterpene yield was 1.13%?RSD=0.89%?.V_C was used as positive control to investigate the effect of total diterpenoids of P.macrophyllus on DPPH radical?DPPH·?,superoxide anion radical?O₂^-·?,hydroxyl radical?·OH?free radical scavenging ability.The results showed that the total diterpenoids of Robinia pseudoacacia had good scavenging effect on three kinds of free radicals,and the scavenging ability was dose-dependent with the mass concentration within the experimental design range.When the total diterpene concentration is 0.59mg/mL,its effect on DPPH·,O₂^-·and·OH the maximum scavenging rates of radicals were 81.71%,66.27%and 38.60%respectively.The extraction method is used to extract the effective components in P.macrophyllus with methanol as solvent,then gradient extraction with different polar solvents such as petroleum ether,chloroform,ethyl acetate,n-butanol and the like is sequentially used to obtain four polar parts,and modern separation technologies such as silica gel column chromatography,sephadex LH-20 column chromatography,preparative thin layer chromatography?PTLC?,semi-preparative high performance liquid chromatography?RPLC?and recrystallization are used to separate two compounds from the ethyl acetate and chloroform extraction parts.The structures of the compounds were identified by modern spectral analysis techniques such as ultraviolet spectroscopy?UV?,nuclear magnetic resonance spectroscopy(¹H-NMR,¹³C-NMR,HMQC,HMBC),EI-MS etc.The confirmed compounds were totarol?1?,4?-carbosy-19-nortotarol?2?,1-deoxy-2?-hydroxy-nagilactone A?3?,podocarpus flavone A?4?,bilobetin?5?,?-sitosterol?6?,tridecanoic acid?7?.Compounds 1,3,4,5 and 7 were isolated from P.macrophyllus for the first time.L-dopa was used as the substrate to investigate the inhibitory effect of total diterpenoids and monomer compounds of P.macrophyllus on tyrosinase.They have different degrees of inhibition on tyrosinase,and the inhibitory effects were totarol>bilobetin>total diterpene>4?-carboxy-19-nortotarol>?-sitosterol.Kinetic studies were carried out on totarol,which showed the best inhibitory activity,and the mechanism type of its inhibitory effect on polyphenol oxidase was determined by using Lineweaver-Burk double reciprocal mapping method.The results show that totarol is a reversible mixed inhibitor,with the michaelis constant K_m of 0.33 mg/mL,the inhibition constant K_I of0.44 mg/mL and K_IS of 0.85mg/mL.