Development of halal and natural skin-lightening topical formulation using hydroxychavicol-rich fraction from the leaves of Piper betle and squid chitosan as one of the excipients

Abstract

Skin-lightening products in the market have raised a serious concern in terms of the skin-lightening agents and non-natural excipients used in the formulations. The concern includes the halal and natural status of the product. Two natural ingredients (hydroxychavicol (HC) and squid chitosan) were used in this study of the developed formulation. HC is one of the major compounds in the leaves of Piper betle Linn. and may possess a skin-lightening effect. Squid chitosan is a nontoxic substance that may act as a carrier for a skin formulation. This study aimed to screen for antioxidant and antityrosinase activities of crude betel leaf extract, HC-rich fraction, and pure HC from the leaves of P. betle, to develop a halal and natural topical formulation using HC as lightening agent and squid chitosan as one of the excipients, and to assess the skin permeation of HC using static Franz diffusion cells and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Initially, fresh P. betle leaves were dried and pulverised into betel leaf powder and macerated in dichloromethane (DCM) solvent to obtain a crude betel leaf extract. The DCM extract was later column chromatographed to obtain HC-rich fraction and pure HC. The extraction process yielded 49 g of crude betel leaf extract, 5 g of HC-rich fraction, and 27 mg of pure HC. The pure HC was authenticated by nuclear magnetic resonance (NMR) spectroscopy. The crude extract, HC-rich fraction, and pure HC were screened for antioxidant and antityrosinase activities. HC-rich fraction was found to possess the highest antioxidant and antityrosinase activities compared to the crude betel leaf extract and pure HC. In developing a halal and natural skin-lightening topical formulation, natural chitosan was extracted from squid pens prior the preparation of the formulation. Natural chitosan was successfully extracted from the squid pens with 31% of yield and characterised using ATR-FTIR spectroscopy by comparing the infrared spectra of the produced chitosan to a commercial chitosan. The natural chitosan was later converted into chitosan acetate to make it a water-soluble excipient and later incorporated into the formulations. The developed topical formulations were prepared into two sets of oil-in-water (O/W) formulations with and without chitosan as carrier for HC. Each set consisted of three formulations differentiated by three kinds of oil that were 4% of sweet almond oil, 2% of jojoba oil, and 4% of olive oil. 0.005% of HC-rich fraction was incorporated into all formulations and 0.4% chitosan acetate was incorporated into the formulations with chitosan. The stability of the formulations was investigated by using fundamental accelerated stability testing for one month and kept in a stability chamber with 40°C and 75% relative humidity. The formulations were characterised in terms of their physical properties, microbial limits, pH, HPLC assay, particle size, zeta potential, and rheological properties. Interestingly, the formulations with chitosan using 4% of olive oil were found to be stable throughout the test. In skin permeation studies, all of the formulations were applied in finite doses on excised human skins and monitored for 24 hr in Franz diffusion study. The skins were later tape stripped. ATR-FTIR spectroscopy was performed to support the evidences of HC permeation and the efficacy of chitosan as carrier. The study found that HC permeated successfully into the skin and no remaining of HC was found on the surface of stratum corneum in tape strip test. Finally, ATR-FTIR spectroscopy proved the efficacy of chitosan in the formulations. Hence, a stable halal and natural skin-lightening topical formulation was successfully produced.