Piperine

Cell lines: three tumorigenic strains and one non-tumorigenic strain were used. The strains are from the American Tissue Cell Culture (ATCC): squamous cell carcinoma of the cervix (SiHa), adenocarcinoma of the cervix (HeLa) and squamous cell carcinoma of the cervix (CaSKi). The tumorigenic lineage studied was the keratinocyte cell line derived from human skin (HaCaT).
Piperine: the compound Piperine (Sigma-Aldrich, St. Louis, MO, USA) was used at a concentration of 25, 50, 100, 150, 200, 250, and 300 μM, diluted in DMSO at concentrations below 0.85% in the tests of cell viability and proliferation and in subsequent assays at concentrations below 0.5%, concentrations that are not toxic to the cells studied [78 (link)].

All the plant parts were shed dried and powdered used grinder separately, passed through a 80-mesh sieve. For extraction 1-g dried powder of each sample were weighed accurately and macerated with methanol for 24 h in a shaker (Lab X incubator shaker) in room temperature. The extracts were filtered via Whatman filter paper (grade-1, 90 mm), and a rotary evaporator was used to evaporate the solvent. Each final extract was weighed, redissolved in methanol (10 mg/mL), and kept in a refrigerator at 4 C for further HPTLC analysis. A precise 5 mg of reference standard piperine (Sigma Aldrich, India) was weighed out and dissolved in 5 mL of methanol to create the standard piperine solution.

Midazolam (Exir Lorestan, Iran), EDTA (Sigma Chemical Co. USA), piperine (Merck, Germany), n-hexan, HPLC grade methanol, isoamyl alcohol and HCl (Merck, Germany) and deionized water were used. Standard form of midazolam and diazepam as base (used as internal standard) were prepared from Dr. Abidi Pharmaceuitical Company (Tehran, Iran).

Piperine (99.5%) and solvents including ethanol (99%), methanol (99.9%), dichloromethane (99.9%), chloroform (99%), acetic acid (99.8%), acetonitrile (99.8%), ethyl acetate (99.5%), n-butanol (99.5%), n-hexane (99.5%) and silica gel (60G, 0.043–0.063 mm particle size), sephadex LH-20 (99.8%) were collected from Merck chemicals. AgNO₃ (99.8%), NaOH (99.5%), Hg(NO₃)₂ (99.9%) at analytical grade were also purchased from Merck chemicals. Fresh P. chaudocanum stems were collected in Son La Province, Vietnam in March 2021, and then washed with deionized water, dried at 50°C, and crushed with a grinder to change into powder form.

Standard compounds, myristicin, plumbagin, 6-gingerol, 6-shogaol, were purchased from Sigma-Aldrich, USA, and piperine was purchased from Merck, Thailand. HPLC-grade water, methanol, and acetonitrile were purchased from Labscan, Thailand.
Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), penicillin streptomycin (P/S), and 0.5% trypsin-EDTA were purchased from Gibco BRL Life Technologies (Grand Island, NY, USA). Phosphate buffer saline (PBS) was purchased from Amresco (Ohio, USA). Dimethyl sulfoxide (DMSO) was purchased from Fluka (Munich, Germany). Lipopolysaccharide (LPS) and 3-(4,5-dimethyl-2-thiazolyl)-2,5 diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Prostaglandin E2 EIA kit monoclonal was purchased from Cayman Chemical Company (Michigan, USA).
Male Sprague-Dawley rats were obtained from National Laboratory Animal Center, Nakorn Pathom, Thailand. They were kept in a room maintained under environmental conditions of 25 ± 1°C and 12 hr dark-light cycle with free access to water and food. Rats were kept in the experimental facility for 1 week to allow them to be acclimated prior to the experiments. The Animal Ethics Committee of Faculty of Medicine, Thammasat University, Thailand, approved the experimental protocols (no. 0002/2008). After the experiments, all animals were sacrificed.