การพัฒนาตัวพาอนุภาคนาโนไขมันที่บรรจุสารสกัดดอกดาวเรืองเพื่อยับยั้งฤทธิ์เอนไซม์ไทโรซิเนส

Abstract

The purpose of this study was to develop nanostructured lipid carrier (NLC) loaded with whitening agent from marigold flowers (Tagetes erecta). The dried flowers were powdered and extracted by Soxhlet’s apparatus with n-hexane, ethyl acetate and ethanol, respectively to obtain hexane extract (HE, %yield = 4.45±0.16), ethyl acetate extract (EE, %yield = 10.26±1.03) and ethanol extract (AE, %yield = 22.83±1.76). This study showed that EE had the highest total flavonoid content (287.93±0.19 mg RE/g of dry extract) and the highest anti-tyrosinase inhibitory activity (IC50=261.20±1.03 µg/ml) among all crude extracts. The EE was chosen to continuously separate by Vacuum Column Chromatography (VCC) technique with gradient elution (100% hexane to 30% MeOH/EtOAc) to obtain 14 fractions (F1 to F14). The fraction 8 (F8) showed the highest flavonoid content (475.56±0.38 mgRE/g of dry extract) and the highest mushroom tyrosinase inhibitory activity (IC50= 50.73±0.56 µg/ml) when compared with other fractions. F8 showed mushroom tyrosinase inhibitory activity more than the reference substances: beta-arbutin 10.57 times, vitamin C 1.66 times, pomegranate extract 3.25 times, patulatin 1.87 times, quercetagetin 1.40 times and the EE 5.15 times. The EE and F8 were then selected to develop into the Nanostructured Lipid Carriers (NLC) by the hot high pressure homogenization technique. The two of NLC base namely A and B formulas were the most suitable for further development. The 1.0%w/w of EE and the 0.2%w/w of F8 were loaded into both of A and B formulas. All loaded NLCs had the average of particle size during 125-150 nm, the average of poly-dispersion index during 0.19-0.22, the average of Zeta-potential -19.46 to -26.68 mV and the average % entrapment during 76.55-88.10%. Moreover, all loaded NLCs could enhanced the chemical stability of quercetagetin, the main compound contained in the marigold flower extracts, and also remained the tyrosinase inhibitory activity after stored in heating-cooling cycles (6 cycles) and 3 months at 4ºC, 45 ºC, light-room temperature (LRT) and dark-room temperature (DRT) conditions. Franz diffusion cell was used for release study and found that the entrapped extract in NLCs could be well released from NLCs. This releasing pattern was matrix from as the initiation (at first 12 hrs) was burst release and after that (at 12-24 hrs) was sustained manner.