Skin Rejuvenation Efficacy and Safety Evaluation of Kaempferia parviflora Standardized Extract (BG100) in Human 3D Skin Models and Clinical Trial

Abstract

Polymethoxyflavones from Kaempferia parviflora rhizomes have been shown to effectively combat aging in skin cells and tissues by inhibiting senescence, reducing oxidative stress, and enhancing skin structure and function. This study assessed the anti-aging effects and safety of standardized K. parviflora extract (BG100), enriched with polymethoxyflavones including 5,7-dimethoxyflavone, 5,7,4'-trimethoxyflavone, 3,5,7,3',4'-pentamethoxyflavone, 3,5,7-trimethoxyflavone, and 3,5,7,4'-tetramethoxyflavone. We evaluated BG100's impact on skin rejuvenation and antioxidant properties using photoaged human 3D full-thickness skin models. The potential for skin irritation and sensitization was also assessed through studies on reconstructed human epidermis and clinical trials. Additionally, in vitro genotoxicity testing was performed following OECD guidelines. Results indicate that BG100 promotes collagen and hyaluronic acid production, reduces oxidative stress, and minimizes DNA damage in photoaged full-thickness 3D skin models. Furthermore, it exhibited non-irritating and non-sensitizing properties, as supported by tests on reconstructed human epidermis and clinical settings. BG100 also passed in vitro genotoxicity tests, adhering to OECD guidelines. These results underscore BG100's potential as a highly effective and safe, natural anti-aging agent, suitable for inclusion in cosmeceutical and nutraceutical products aimed at promoting skin rejuvenation.

Keywords: 3D skin model; Kaempferia parviflora standardized extract; anti-aging; clinical trial; polymethoxyflavones.

Figure 1

BG100 extract enhances collagen type I expression in UV-exposed human full-thickness 3D skin tissues. (a) Representative images of 3D skin tissues exposed or not exposed to UVA and UVB for 5 days, followed by topical application of 2% ethyl ascorbic acid in water, water, 0.5% retinol in DMSO, 0.5% DMSO, 0.1% and 0.2% BG100 extracts in ethanol, or 1% ethanol for 6 days, depicting collagen type I (green) and DAPI nuclear staining (blue). (b) Quantification of collagen type I intensity (a) expressed as integrated density. Two skin tissues were employed for each treatment, and 3 sections per tissue were imaged (6–8 images/section). * p < 0.05; **** p < 0.0001 compared to the UV-exposed negative control.

Figure 2

BG100 extract enhances hyaluronic acid secretion in UV-exposed human full-thickness 3D skin tissues. Hyaluronic acid secretion was measured in the culture medium of human full-thickness 3D skin tissues, which were either exposed or not exposed to UVA and UVB radiation for 5 days, and topically treated with 2% ethyl ascorbic acid in water, water, 0.5% retinol in DMSO, 0.5% DMSO, 0.1% and 0.2% BG100 extracts in ethanol, or 1% ethanol, for 6 days. Data are based on duplicate measurements from two skin tissues for each treatment. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, compared to the UV-exposed negative control.

Figure 3

The antioxidant effect of BG100 extract demonstrated by inhibiting Reactive Oxygen Species (ROS) in UV-exposed human full-thickness 3D skin tissues. ROS level was measured in the culture media of human full-thickness 3D skin tissues that were exposed or not exposed to UVA and UVB for 5 days and topically treated with 2% ethyl ascorbic acid in water, water, 0.5% retinol in DMSO, 0.5% DMSO, 0.1% and 0.2% BG100 extracts in ethanol, or 1% ethanol for 6 days. Data are based on duplicate measurements from two skin tissues per treatment. **** p < 0.0001, compared to the UV-exposed negative control.

Figure 4

BG100 extract protects against DNA damage in UV-exposed human full-thickness 3D skin tissues. 8-Oxo-2′-deoxyguanosine (8-OHdG) was measured in the culture medium of human full-thickness 3D skin tissues that were exposed or not exposed to UVA and UVB for 5 days and topically treated with 0.5% retinol in DMSO, 2% ethyl ascorbic acid in water, 0.1% and 0.2% BG100 extracts in ethanol, 0.5% DMSO, 1% ethanol, or ethanol alone for 6 days. Data are based on duplicate measurements from two skin tissues per each treatment. ** p < 0.01; **** p < 0.0001, compared to the UV-exposed negative control.

Figure 5

Safety evaluation of BG100 extract on in vitro reconstructed 3D human skin tissues (OECD TG 439). (a) MTT interference test performed by adding 1 mg/mL of MTT solution with 0.2% BG100 extract, KOH (positive control), or PBS (negative control). (b) Percentage of relative cell viability of the reconstructed human epidermis tissues treated with 0.2% BG100 extract, 5% SDS (positive control), or PBS (negative control) for 1 h and continued incubation for 24 h. (c) IL-1α expression in tissue culture medium of reconstructed human epidermis tissues exposed to the same treatments as (b). Data are based on duplicate measurements from three skin tissues per each treatment. **** p < 0.0001, compared to the negative control.

Figure 6

BG100 extract exhibits non-genotoxic effects on V79-4 cells without S9 fraction following 4-h and 24-h treatments. (a) Micronucleus (MN) frequency and (b) cytokinesis-block proliferation index (CBPI) values were calculated. V79-4 cells were exposed to 50, 100, 200, and 400 µg/mL BG100 extract, 1.25 µg/mL mitomycin C (MMC; positive control), or culture medium (negative control) for 4 or 24 h. Subsequently, cytochalasin B was added and incubation continued for an additional 18–20 h. Data are presented as mean ± SEM from a single independent experiment conducted in triplicate. * p < 0.05, compared to the negative control.

Figure 7

BG100 extract is non-genotoxic to V79-4 cells at concentrations of 50, 100, 200, and 400 µg/mL in the presence of S9 fraction after 4-h treatment. (a) MN frequency and (b) the CBPI value were calculated. V79-4 cells were treated with 50, 100, 200, and 400 µg/mL BG100 extracts, 5 µg/mL benzo[a]pyrene (BP; positive control), or culture medium (negative control) for 4 h. Cells were treated with S9 fraction for 4 h, then cytochalasin B was added and incubation continued for 18–20 h. MN frequency and CBPI were calculated and reported as Mean ± SEM. Results were performed from one independent experiment with triplicate measurements. * p < 0.05, compared to the negative control.

Figure 8

Flow diagram illustrating the clinical study focused on assessing the skin sensitization and irritation potential of BG100 extract.