The Combination of Pterocarpus marsupium Bark Extract, Pinus strobus Bark Extract, and Ascorbyl Tetraisopalmitate Inhibits Melanogenesis via Nicotinamide Nucleotide Transhydrogenase Activation

ABSTRACT Background Excessive ultraviolet exposure triggers skin pigment deposition, potentially leading to conditions such as melasma, freckles, and post‐inflammatory hyperpigmentation. The skin of individuals with these dermatological conditions exhibits reduced levels of nicotinamide nucleotide transhydrogenase (NNT). While many seek skin‐whitening products to address pigmentation, existing options often fail to achieve optimal efficacy. To accomplish a synergistic whitening effect, we developed PPV, a composite formulation incorporating three depigmenting agents: Pterocarpus marsupium bark extract (PMBE), Pinus strobus bark extract (PSBE), and ascorbyl tetraisopalmitate (VC‐IP). Aims This study aimed to investigate the effects of PPV on skin whitening and elucidate its mechanisms. Methods After administering PPV to B16F10 melanoma cells, we measured melanin content and gene expression of TYR , TRP‐1 , and TRP‐2 using a microplate reader and qPCR. Western blotting was used to assess p‐CREB, CREB, MITF, and NNT expression. The Jin's formula was used to evaluate the combined effect of the three components of PPV on NNT at the genetic level. Oxidative stress levels were evaluated with ROS, GSH/GSSG, and NADP + /NADPH assay kits, along with confocal microscopy. Transmission electron microscopy and ELISA were employed to quantify the eumelanin/pheomelanin ratio and melanosome maturation. Finally, immunodetection was performed to assess epidermal basal pigmentation, tyrosinase, and NNT expression in a human ex vivo skin model. Results PPV significantly influenced melanin production in melanocytes and human ex vivo skin. This effect was due to the inhibitory effect of PPV on the CREB‐MITF signaling pathway and the synergistic activation effect of PMBE, PSBE, and VC‐IP on the NNT gene expression in B16F10 cells. Activation of NNT led to a reduction in ROS levels and the NADP + /NADPH ratio, ultimately inhibiting melanogenesis, tyrosinase activity, and melanosome maturation in B16F10 cells. Furthermore, PPV treatment reduced pigmentation and increased NNT expression in the aged ex vivo skin model. Conclusion PPV, synthesized from PMBE, PSBE, and VC‐IP, exhibited an anti‐melanogenic effect. This effect was attributed to the inhibition of CREB‐MITF‐mediated melanogenesis and the regulation of oxidative stress by NNT. The findings suggest PPV as a potential therapeutic agent for skin pigmentation disorders.