Presence of Cholesterol in Non-Animal Organisms: Identification and Quantification of Cholesterol in Crude Seed Oil from Perilla frutescens and Dehydrated Pyropia tenera

Abstract

Studies have reported that cholesterol, a molecule found mainly in animals, is also present in some plants and algae. This study aimed to determine whether cholesterol exists in three dehydrated algae species, namely, Pyropia tenera, Saccharina japonica, and Undaria pinnatifida, and in one plant species, namely, Perilla frutescens (four perilla seed oil samples were analyzed). These species were chosen for investigation because they are common ingredients in East Asian cuisine. Gas chromatography-flame ionization detection (GC-FID) analysis found that cholesterol was present in P. tenera (14.6 mg/100 g) and in all four perilla seed oil samples (0.3-0.5 mg/100 g). High-performance liquid chromatography with evaporative light-scattering detection (HPLC-ELSD) also demonstrated that cholesterol was present in P. tenera (14.2 mg/100 g) and allowed the separation of cholesterol from its isomer lathosterol. However, cholesterol could not be detected by HPLC-ELSD in the perilla seed oil samples, most likely because it is only present in trace amounts. Moreover, liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed the presence of cholesterol in both P. tenera and perilla seed oil. MRM results further suggested that lathosterol (a precursor of cholesterol) was present in P. tenera.

Keywords: GC-FID; HPLC-ELSD; LC-MS/MS; algae; cholesterol; lathosterol; perilla seed oil.

Figure 1

Scheme of the experimental design.

Figure 2

Gas chromatograph-flame ionization detector (GC-FID) chromatogram of a mixed standard (cholesterol, lathosterol, and α-tocopherol) and samples. (A), mixed standard; (B), Pyropia tenera; (C), perilla seed oil; (C *), cholesterol peak of four perilla seed oil samples; (D), Saccharina japonica; (E), Undaria pinnatifida. Peak identification: 1, 5-α-cholestane (IS); 2, α-tocopherol; 3, cholesterol; 4, lathosterol; 5, β-sitosterol a-b, unknown peak; c, unknown peak (RRT: 1.35) around lathosterol and cholesterol. Relative retention time (RRT): value obtained by dividing the compound retention time by the internal standard retention time.

Figure 3

Chromatograms of mixed standards and samples using high-performance liquid chromatography-evaporative light-scattering detection (HPLC-ELSD). (A), mixed standard (a, lathosterol standard 0.005 mg/mL and cholesterol standard 0.01 mg/mL; b, lathosterol standard 0.0025 mg/mL and cholesterol standard 0.005 mg/mL). (B), Pyropia tenera; (C), perilla seed oil. Peak identification: 1, lathosterol; 2, cholesterol.

Figure 4

Multiple reaction monitoring (MRM) spectra of cholesterol standard (a), lathosterol standard (b), Pyropia tenera (c), perilla seed oil (d). Enlarged MRM spectrum of each peak 1, 2, 4, and 6 (e).

Figure 5

Suggested cholesterol synthesis pathway in algae and plants. This Figure is adapted from Sonawane et al. [21], Yoshida et al. [39], and Belcour et al. [40].