Structural Characteristics and Hypolipidemic Activity of Theabrownins from Dark Tea Fermented by Single Species Eurotium cristatum PW-1

Abstract

Recently, studies on theabrownins (TBs), the main bioactive polymeric pigments found in dark tea, have received increasing attention for its health effects. Thus far, information on their structural characteristics is unclear. In the present study, theabrownins were isolated from single species Eurotium cristatum PW-1-fermented loose tea and their structural and hypolipidemic characteristics were studied for the first time. The theabrownins were fractionated by their molecular weights and were then analyzed. Ultraviolet-visible spectrophotometry (UV-Vis) and Flourier transformation infrared spectroscopy (FT-IR) showed that they were polymerized phenolic substances containing abundant hydroxy and carboxyl groups. All theabrownin samples exhibited hypolipidemic activity in high-fat zebrafish; among which TBs-10-30k sample, decreased lipid level in high-fat zebrafish to 51.57% at 1000 μg/mL, was most effective. It was found that TBs-10-30k was a type of amorphous and thermostable polymer with slice shape and smooth surface under scanning electron microscope (SEM). Atomic force microscope (AFM) analysis showed that it had island-like structure because of aggregation of theabrownin molecules. Pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) analysis further showed that the main pyrolytic products of TBs-10-30k were hexadecanoic acid (33.72%), phenol (14.90%), and eicosane (12.95%), indicating TBs-10-30k was mainly composed of phenols, lipids, saccharides, and proteins. These results not only facilitate subsequent identification of theabrownins, but also provide insights into the applications of theabrownins in functional foods.

Keywords: E. cristatum fermented loose tea; fractionated; hypolipidemic activity; structural characteristics; theabrownins.

Figure 1

Ultraviolet–visible spectra (A) and Flourier transformation infrared spectra (B) of theabrownins (TBs) and fractionated theabrownin samples with different molecular weights. TBs-LT3k, TBs-3-10k, TBs-10-30k, and TBs-GT30k represented the theabrownin samples with molecular weights: <3 kDa, 3–10 kDa, 10–30 kDa, and >30 kDa, respectively.

Figure 2

Hypolipidemic effect of theabrownins (TBs) and fractionated theabrownin samples with different molecular weights in high-fat-induced obesity zebrafish. (A) Experimental outline of zebrafish model. (B) Zebrafish stained with oil red O and visualized under a microscope magnified 30 times. (C–E) Relative lipid accumulation in zebrafish treated with different concentrations of theabrownins. Data were expressed as mean ± SEM (n = 6). *p-value < 0.05 and #p-value < 0.001 compared to high-fat diet (HFD) group. HFD, high-fat diet-induced obesity zebrafish. DMSO, vehicle control at 0.1% (v/v). Simvastatin, positive control at 0.06 μM. TBs-LT3k, TBs-3-10k, and TBs-10-30k represented the fractionated theabrownin samples with molecular weights: <3 kDa, 3–10 kDa, and 10–30 kDa, respectively. Scale bar, 500 μm.

Figure 3

Thermogravimetry/Differential thermogravimetry analysis (A) and differential scanning calorimetry (B) analysis results of TBs-10-30k.

Figure 4

X-ray diffraction pattern of TBs-10-30k.

Figure 5

Morphological characteristics of TBs-10-30k. (A) Images magnified by scanning electron microscopy at 500 and 1000 times, respectively. (B) Planar images and corresponding three-dimensional images of TBs-10-30k obtained by atomic force microscopy. The scanning range is 5 × 5 μm and 1 × 1 μm, respectively.