Margaric acid

Progesterone injection was purchased from Xianju Pharma (Taizhou, China). Pentobarbital was obtained from Xiya Reagent (Chengdu, China). Ultrapure water was produced by a Milli-Q plus water purification system (Milford, USA). N,O-bis(trimethylsilyl)-trifluoroacetamide-trimethylchlorosilane (BSTFA-TMCS) (99 : 1, v/v), urease, margaric acid, tropic acid, methoxylamine hydrochloride, pyridine, and metabolite standards were purchased from Sigma-Aldrich (St. Louis, USA). Absorbable gelatin sponge was purchased from Jinling Pharmaceutical Co. (Nanjing, China).
PSF was purchased from Tianxiang Co. (Yueyang, China) and identified by Professor Zhuxin Wang (Hunan university of Chinese medicine, Changsha, China). A voucher specimen (number HHBJC20140913) is deposited in the Key Laboratory of Hunan Province for Traditional Chinese Medicine in Obstetrics & Gynecology Research (the Maternal and Child Health Hospital of Hunan Province, Changsha, China). Referring to previous method [25 (link)], a 500 g of PSF was two times extracted with 5 L of hot ethanol for 1 hour and then the two extracts were filtrated. The filtrate was dried in vacuo to yield 63.9 g of the PSF extract.

Lauric acid (98%), linoleic acid (≥ 99%), margaric acid (≥ 98%), myristic acid (≥ 99%), oleic acid (≥ 99%), palmitic acid (≥ 99%), and stearic acid (≥ 98.5%) as well as formic acid (98–100%), HPLC plus grade acetone, HPLC gradient grade acetonitrile (ACN), HPLC grade ethanol (EtOH), and HPLC gradient grade methanol (MeOH) were purchased from Sigma Aldrich (Steinheim, Germany). Ultra-pure deionized (DI) water was delivered by a Milli-Q® system (Merck, Darmstadt, Germany).

Sulfuric acid, methanol, butylated hydroxytoluene (BHT), toluene, sodium chloride, anhydrous sodium sulfate, and diethyl ether were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Hexyl hexanoate, fatty acid methyl ester (FAME), and alkane reference standards were purchased from J&K Chemical Ltd. (Shanghai, China). Margaric acid and ethyl caprate were purchased from Sigma-Aldrich (Steinheim, Germany).
Thirty-seven FAME reference standards with concentrations between 100 ng/mL and 200 ng/mL (C4–C24) were mixed, while FAME reference standards with carbon numbers of C16-C18 were prepared individually, and the purity was 98–99%. The alkane reference standards (C4–C40) were dissolved in chloroform with a concentration of 500 μg/mL.

Analytical-grade reagents and solvents, i.e., Folin–Ciocalteu reagent from Sigma-Aldrich (Saint Louis, MO, USA), anhydrous sodium sulfate (Na₂SO₄), chloroform, diethyl ether, ethanol (99.9% purity), formic acid, methanol, potassium hydroxide (KOH), sodium carbonate (Na₂CO₃), sulfuric acid (H₂SO₄), and zinc purchased from Chempur (Piekary Śląskie, Poland) were used. Chromatography-grade solvents, i.e., dichloromethane, ethanol, methanol, n-hexane, heptane, methyl tert-butyl ether, iso-propanol, pyridine, and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Analytical standards such as 5α-cholestane, margaric acid, β-apo-8’-carotenal, D-catechin, phenolic acids (p-coumaric acid (p-CA), dihydroferulic acid (DHFA), sinapic acid (SA), vanillic acid (VA)), and 4-vinylguaiacol (4-VG) were purchased from Sigma-Aldrich (Saint Louis, MO, USA), while tocopherols (α-, γ-, and δ-tocopherol) were purchased from Calbiochem (Nottingham, UK). Deionized water was obtained from HLP 5 deionizer (Hydrolab, Gdańsk, Poland).