Steroid 5-alpha reductase inhibitors, physicochemical properties, and preformulation studies of teak leaf extract

Abstract

The enzyme steroid 5α-reductase plays a pivotal role in the conversion of testosterone to dihydrotestosterone, a process linked to various androgen-dependent disorders. In the previous study, our research group explored the biological activities of Tectona grandis L.f. extracts, particularly their potential for addressing hair loss concerns. Notably, the hexane extract of T. grandis leaves exhibited promise as a 5α-reductase inhibitor. The objectives of the current study were therefore to purify and identify the 5α-reductase inhibitors from the T. grandis leaf-hexane extract. These isolated compounds were then used as bioactive compounds for the standardization of the extract. The physicochemical properties and stability profiles of extracts were also investigated. The study was extended to examine the skin penetration of extracts and their bioactive constituents, which will provide necessary information for the future formulation of products containing T. grandis leaves.

Three terpenoid compounds isolated from T. grandis leaves, namely (+)-eperua-8,13-dien-15-oic acid (1), (+)-eperua-7,13-dien-15-oic acid (2), and lupeol (3), were successfully identified by spectroscopic analysis. Their 5α-reductase inhibitory activity was assessed, revealing that compound 3 had weak inhibitory -activity while 1 and 2 exhibited potent inhibition. An HPLC method was developed for the quantitative determination of two potent inhibitors (1 and 2) and quality control of T. grandis leaf extracts. Compounds 1 and 2 displayed higher content in the ethanolic extract than hexane extract suggesting that ethanol is a more preferable solvent for extraction. The quality control and extraction methods are beneficial for the continued development of T. grandis leaf extract as a hair loss treatment ingredient.

The appropriate vehicle for the in-vitro skin penetration study was determined using the shake flask method. Compounds 1 and 2 have poor solubility in distilled water while their solubility in HEPES buffer with 2% w/v of Tween20 was significantly greater. These findings suggest that the HEPES buffer may be an appropriate vehicle for further skin penetration study. The partition coefficients of 1 and 2 were hydrophobic compounds. As well, other physicochemical properties were also reported. The study of physicochemical properties provides information for designing and developing an appropriate formulation for a more effective assessment of bioactive compounds in pre-formulation studies.

Furthermore, there is currently no information in the literature on a pre-formulation study of two 5α-reductase inhibitors (1 and 2) in T. grandis extract, and their chemical stability is unknown. Therefore, this research aimed to determine how pH, temperature, and light affected the stability of 1 and 2 in crude ethanolic extract and ethanolic extract in PG with PEG-40 as a solubilizer. Moreover, these two extracts were also tested in buffered solution forms. Their residual concentrations of 1 and 2 were analyzed using the HPLC method. Compounds 1 and 2 were stable in acidic environments. Their degradations were extremely sensitive to light, particularly the degradation of 1 in the buffered solution form of ethanolic extract in PG. In the buffered solution form of ethanolic extract, both marker compounds exhibited rapid degradation under high temperatures. However, the addition of PG effectively reduced their degradation, particularly when protected from light. The calculated shelf-life of 1 and 2 was 1.48 years. Therefore, the extracts should be stored in a prepared extract form or otherwise in a slightly acidic environment with light protection. Additionally, the in-vitro skin penetration of 1 and 2 in both ethanolic extract and ethanolic extract in PG was evaluated using Franz diffusion cells. After 24 h, 1 demonstrated notably greater penetration in both extracts, showing significantly higher cumulative amounts (%) than 2. These observations indicate that the higher hydrophobicity of 1 facilitated its more efficient penetration into the stratum corneum. These studies provide valuable information that will aid in the further development of T. grandis extract-containing hair loss treatments.

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