| Platycodon gradiflorum is the dried root of Platycodon gradiflorum（jacq.）a.dc.,a plant of Citridae,which is recorded in Chinese Pharmacopoeia（2020 edition）,and the earliest record can be traced back to Shennong Medica Classic.Platycodon grandiflorum is a Chinese medicinal material with the same origin of medicine and food.It has the pharmacological effects of antitussive expectorant,anti-inflammatory and bacteriostatic,anti-tumor,anti-oxidation,whitening and skin care,immune regulation,etc.In recent years,Platycodon grandiflorum is widely used as the raw material of food,medicine and cosmetics.Objective:In this paper,Platycodon grandiflorum was used as the main body of the study.The modern extraction and separation technology of traditional Chinese medicine combined with pharmacological tracking method was used to screen and find the active parts with whitening effect at the molecular level.The chemical composition and fingerprint analysis of the active fraction were carried out.The mechanism of whitening activity was studied at the cellular level.This study laid a foundation for further exploitation and utilization of platycodon grandiflorum,and also provided a theoretical basis for the development of natural whitening cosmetics.Methods:1.Extraction and separation of whitening active part:With oxygen free radical,tyrosinase and hyaluronidase as targets,antioxidant and enzyme inhibitory activities as indexes,the activity of different extracts with whitening effect reported in literature was screened by pharmacodynamic tracking method,and the whitening active extracts were obtained.Single factor investigation and response surface design were used to optimize the extraction process.The modern extraction technology of traditional Chinese medicine and classical chemical separation technology were applied to separate and purify the best active extract,according to the results of antioxidant and enzyme inhibitory activities,the best active part of Platycodon grandiflorum（PGE-ACT-WT）was obtained.2.Chemical composition analysis of whitening active parts:Q-Orbitrap high resolution liquid-mass spectrometry and other modern chemical composition analysis techniques were used to comprehensively identify and analyze the active components and chemical components in PGE-ACT-WT.3.Fingerprint study of whitening active parts:Based on the active components obtained,the HPLC fingerprint chromatogram was established by using the HPLC-evaporative light scattering detection method and the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint.The chemical pattern recognition analysis of PGE-ACT-WT was carried out by combining cluster analysis and principal component analysis.4.Study on the mechanism of whitening active parts:By B16F10 melanoma cells in mice,HepG2 human liver cells,the original 264.7 macrophages as an object,to tyrosinase,melanin,oxidation index targets,inflammation factors,at the cellular level research PGE-ACT-WT enzyme inhibition,melanin inhibiting,antioxidant and anti-inflammatory activity,from the three aspects of comprehensive investigate the whitening effect mechanism of PGE-ACT-WT.Results:1.Extraction and separation of whitening active part:Using pharmacodynamic tracking method,taking antioxidant activity,tyrosinase inhibitory activity and hyaluronidase inhibitory activity as indexes,11 extracts obtained by the designed three extraction processes were screened.Finally,extract 11（95%alcohol extract PGE-E5）was obtained as the whitening active extract of Platycodon grandiflorum.Single factor investigation and response surface design experiments were used to optimize the extraction process parameters,and the best whitening active extract of Platycodon grandiflorum（PGE-ME5）was obtained.The results showed that the extraction times were 3 times,the extraction time was 1 h,and the amount of solvent was 8 times.The best whitening activity isolate 3（70%ethanol eluate PGE-E3）of Platycodon grandiflorum was obtained by activity screening.Through the above extraction and separation process,the best whitening active part of Platycodon grandiflorum（PGE-ACT-WT）was obtained.2.Chemical composition analysis of whitening active parts:Application of Q-Orbitrap fluid of high resolution mass spectrometry identification from the PGE-ACT-WT got 45 compounds,mainly for the saponins class:Platycodonin E,Platycodonin D3,Baicalin,Platycodonin D.Phenolic acids:Arbutin,Chlorogenic acid,Niacin,α-keto acid.Flavonoids:Quercetin,Mignonette,daidzein yuan,baicalin.Phenyl propyl alcohol glycosides:lilacs glycosides.In addition,a large number of saponins,phenolic acids and flavonoids were identified,and the above compounds have been shown to have certain potential whitening activity.3.Fingerprint study of whitening active parts:On the basis of the chemical composition analysis,using high performance liquid chromatography with evaporative light scattering methods,use of traditional Chinese medicine chromatographic fingerprint similarity evaluation system,using more proofreading the teachings established PGE-ACT-WT fingerprint,according to the generation of 19 common peak fingerprint model,by using the method of reference substance than by retention time and chromatographic behavior of comparison,has identified eight chromatographic peak,Arbutin,Syringin,Chlorogenic Acid,Platycodonin E,Platycodonin D3,Baicalin,Platycodonin D,Luteolin,the eight elements are included in the PGE-ACT-WT liquid qualitative analysis results.4.Study on the mechanism of whitening active parts:4.1 Inhibitor of tyrosinase activity and melanin production The experimental results showed that each dose of PGE-ACT-WT could inhibit the intracellular tyrosinase activity and melanin production in B16F10 cells in a dose-dependent manner.The inhibitory activity of tyrosinase in the high-dose PGE-ACT-WT group was significantly higher than that in the arbutin and platycodon D positive control groups.At 48 h after administration,PGE-ACT-WT showed the strongest cell tyrosinase inhibitory activity and melanin production inhibitory activity,but when the administration time increased to 72 h,the activity decreased,indicating that the optimal administration time of PGE was 48 h.4.2 Antioxidant test results showed that the MDA content of supernatant of HepG2 cells decreased in a dose-dependent manner,and the T-AOC ability increased in a dose-dependent manner.The MDA content of the high-dose PGE-ACT-WT group was significantly lower than that of the VC-positive control group,and the T-AOC ability of the PGE-ACT-WT groups was significantly higher than that of the VC-positive control group,indicating a strong antioxidant capacity.4.3 The results of anti-inflammatory experiment showed that each dose of PGE-ACT-WT group decreased the levels of TNF-αand IL-6 pro-inflammatory factors in LPS-stimulated RAW264.7 macrophages in a dose-dependent manner,showing strong anti-inflammatory activity.The above results indicate that the whitening activity of PGE-ACT-WT is mainly through the good inhibition of cell tyrosinase activity,inhibition of cell melanin production activity,antioxidant activity and anti-inflammatory activity,which can achieve skin whitening effect.Conclusion:In this study,modern traditional Chinese medicine extraction and separation technology combined with pharmacodynamic tracking method was used to obtain PGE-ACT-WT.Q-Orbitrap high resolution liquid-mass spectrometry technology was used to determine the composition of pharmacodynamic components of PGE-ACT-WT.The fingerprint of PGE-ACT-WT was established by HPLC-ELSD technology combined with modern analytical method.The mechanism of whitening activity of PGE-ACT-WT was studied at cell level by using modern biological technology,which provided theoretical basis for the development and application of whitening products of Platycodon grandiflorum. |
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