Anti-tyrosinase and antioxidant activities of Morinda Citrifolia fruit extract and development of nanoemulsions

Abstract

The present study focused on anti-tyrosinase and antioxidant activities of Thai Morinda citrifolia fruit extracts as potential ingredient used for nanoemulsions formulation in cosmetic applications. The fruit juice was freeze-dried. The residue was further extracted by ethanol, acetone and ethyl acetate. All of the extracts were determined for their inhibitory activities against tyrosinase enzyme and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. The extract which showed highest activity was further partitioned by hexane and ethyl acetate. The IC50 of partitioning fractions was determined for their activities. The extract with highest activities was selected for further investigations. The cytotoxicity evaluation was determined by MTT assay using keratinocyte (HaCaT cells). The selected extract was incubated at 4°C, 30°C and 40°C for 6 months. The chemical constituents were determined by validated HPLC condition with scopoletin as a marker. The o/w nanoemulsions with small droplet size was produced and investigated for its stability and in vitro permeation profile. As the results of extraction, the ethanol extraction showed the highest antioxidant activity (IC50 =0.234 mg/ml) with comparable anti-tyrosinase activity to other solvent extracts. After partition, ethyl acetate soluble fraction exhibited highest activities with IC50 value of anti-tyrosinase and DPPH assay were 1.201 mg/ml and 0.014 mg/ml, respectively. The extract at concentration of 1.2 mg/ml had no effect on cell viability. The stability determination of the extract showed insignificant reduction of the scopoletin concentration as well as its antioxidant activity against DPPH radicals kept at 4°C and 30°C for 6 months. However, it showed slightly reduction after storage at 40°C for 4 months. This reduction was in an acceptable range. Caprylic/capric triglyceride was chosen as the oil phase, TWEEN®80-SPAN®20 as emulsifiers, and cremophor® RH-40 as solubilizing enhancer for nanoemulsions formulation. The prepared nanoemulsions showed good physical stability under heat-cool stress conditions and kept at 4°C and 30°C over 3 months. The chemical compound, scopoletin, incorporated in nanoemulsions exhibited good stability. The release profile of extract-loaded nanoemulsions was not different compared to the solution of extract. The extract-loaded nanoemulsions showed significantly better permeation through porcine membrane than conventional o/w emulsion.