Pro collagen 1

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin/EDTA, and penicillin/streptomycin were from Gibco (Life Technologies, Grand Island, NY, USA). Antibodies of glyceroaldehyde-3-dehydrogenase (GAPDH) were purchased from Santa Cruz, whereas antibodies for Ac-H3, OPG and RANKL were from GeneTex. Ethidium bromide, agarose and kits for reverse transcription (RT) and polymerase chain reaction (PCR) were purchased from HT Inc., UK. Total RNA isolation kits were from Qiagen Inc. (Santa Clarita, CA, USA). Specific PCR primer sets were synthesized by Genemed Biotechnologies, Inc. (San Francisco, CA, USA). Protein assay kits were obtained from Bio-Rad (Bio-Rad Labs, Hercules, CA, USA). Sodium butyrate was obtained from Sigma (Sigma-Aldrich Company, St. Louis, MO, USA). MG-63 cells were obtained from the American Type Culture Collection (ATCC). OPG, RANKL, pro-collagen I, MMP-2, osteopontin, osteocalcin and osteonectin (SPARC) ELISA kits were from R&D. 8-Isoprostane ELISA kits were from Cayman.

Human primary dermal fibroblasts HDFa (ATCC, Manassas, VA, USA) were seeded in a 24-well plate at a density of 1.5 × 10⁴ cells per well in S10 cultivation media (DMEM supplemented with 1% penicillin (100 U mL⁻¹)–streptomycin (100 μg mL⁻¹) (P/S) and 10% FBS). The cells were incubated for 24 h at +37 °C with 5% CO₂ to allow them to attach and initiate proliferation. Fresh media containing a combination of extracts were added, and the cells were cultivated at +37 °C with 5% CO₂. Media samples were collected for analysis after 24 and 72 h of incubation with the combination. The secretion of pro-collagen I and MMP-1 in the cultivation media was quantified using ELISA immunoassays employing R&D Systems DuoSet Human pro-collagen I alpha 1 and MMP-1 ELISA kits (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s recommendations. Standard dilutions of pro-collagen I (R&D Systems Minneapolis, USA) and MMP-1 (R&D Systems Minneapolis, USA) were utilized to generate the standard curve and calculate the concentrations of both analytes in the cultivation media.