Ellagic acid (EA) is a biologically active phenolic compound widely present in plant tissues such as fruits and nuts, with its structure shown in Figure 1. It is a dimeric derivative of gallic acid. In nature, ellagic acid mainly exists in condensed forms, such as ellagitannins. Ellagic acid is essentially non-toxic and has a significant inhibitory effect on chemically-induced carcinogenesis and various types of cancers, including colon cancer, liver cancer, lung cancer, prostate cancer, breast cancer, lymphoma, and others. Additionally, ellagic acid also exhibits antioxidant, anti-inflammatory, antibacterial, melanin inhibition, and leukemia improvement effects. Due to its excellent biological activity, ellagic acid is widely used in health foods, medicine, pharmaceuticals, cosmetics, and other fields.
Studies have shown that Tea is an important dietary source of ellagitannins and ellagic acid, which are converted from ellagitannins through human gut microbiota Metabolism.
▲ Human Metabolic Pathway
Pu'er ripened tea, a famous Yunnan tea, is a unique microbial-fermented tea made from large-leaf sun-dried Green Tea. The essence of solid-state fermentation in Pu'er ripened tea is primarily the result of moist heat, enzymatic actions, and microbial activities. During the fermentation process of large-leaf green teas, microbial metabolism releases a large amount of heat, and the enzymes secreted react intensely with the endogenous substances in the tea, both of which can promote the decomposition of heat-unstable ellagitannins (such as casuarinin) into ellagic acid.
The establishment of a detection method for ellagic acid content in Pu'er ripened tea can meet the quality control needs of characteristic bioactive components in Pu'er ripened tea and also contribute to the study of the changes in characteristic substances during the fermentation process of Pu'er tea and the metabolic patterns of microorganisms. This provides a methodological basis for the industrial controllable fermentation of Pu'er tea.
01
Materials and Methods
1. Instruments and Equipment
Waters e2695 high-performance liquid chromatograph (HPLC) (Waters Corporation, USA); Waters 2995 PDA (photodiode array detector, Waters Corporation, USA); WD-9415B ultrasonic bath (Beijing Liuyi Instrument Factory); H1850R low-temperature centrifuge (Xiangyi Centrifuge Instrument Co., Ltd.); VORTEX 3 vortex mixer (IKA GmbH, Germany); analytical balance (precision 0.0001 g, OHAUS Corporation, USA); Agilent ZORBAX SB-C18 chromatographic column (250 mm × 4.6 mm, 5 μm, Agilent Technologies, USA).
2. Materials and Reagents
Syringe filters (0.45 μm, nylon 66, Tianjin Jinteng Experimental Equipment Co., Ltd.); acetonitrile, methanol (both HPLC grade, Merck KGaA, Germany); water (ultrapure); phosphoric acid (analytical grade); ellagic acid reference standard (purity ≥ 98%, Shanghai Yuanye Bio-Technology Co., Ltd.).
3. Chromatographic Conditions
Agilent ZORBAX SB-C18 chromatographic column (250 mm × 4.6 mm, 5 μm); 0~20 min, 0.2% phosphoric acid: acetonitrile (85:15, v/v), isocratic elution; flow rate: 1 mL/min; injection volume: 10 μL, column temperature 35 °C; photodiode array detector; detection wavelength 245 nm.
4. Preparation of Standard Solution
A certain amount of ellagic acid reference standard was weighed and dissolved in methanol to prepare a stock solution with a mass concentration of 60 mg/L, which was stored at -4 °C in the dark. Before use, an appropriate amount of the ellagic acid stock solution was accurately transferred and diluted with methanol to prepare working solutions with mass concentrations ranging from 1 to 30 mg/L, which were stored at -4 °C in the dark.
5. Sample Preparation
An appropriate amount (accurate to 0.0001 g) of uniformly ground ripened tea sample was weighed into a 50 mL centrifuge tube, and an appropriate amount of extraction solvent (water, 25% methanol, 50% methanol, 75% methanol, pure methanol) was added so that the material-to-solvent ratio was 1:10, 1:20, 1:30, 1:40, and 1:50. The mixture was ultrasonicated for a certain period (30 min, 45 min, 60 min), centrifuged at 5000 r/min for 10 min, and the supernatant was transferred to a 100 mL volumetric flask. This process was repeated (0 times, 1 time, 2 times, and 3 times, i.e., extracted 1 time, 2 times, 3 times, and 4 times), and the supernatants were combined and diluted to 100 mL with water. After shaking well, the solution was filtered through a 0.45 μm water-phase filter membrane and then prepared for analysis.
02
Results and Analysis
1. Optimization of HPLC Conditions
Using different mobile phases (water-methanol, water-acetonitrile, phosphoric acid solution-methanol, and phosphoric acid solution-acetonitrile, with phosphoric acid concentrations of 0.2%, 0.3%, and 1%), isocratic elution was performed to investigate the impact of different mobile phase types, ratios, and pH values on the separation efficiency of ellagic acid in Pu'er ripened tea. The evaluation focused on aspects such as peak shape, resolution from interfering peaks, etc.
The results showed that when using a water-organic phase system as the mobile phase, the ellagic acid peak was broad and had severe tailing, making it difficult to identify. By adding different concentrations of phosphoric acid to the elution system to optimize the peak shape, the peak width of the ellagic acid peak became narrower, and the peak shape became more symmetrical with slight tailing. However, there was no significant difference in peak shape changes at different phosphoric acid concentrations, so a 0.2% phosphoric acid concentration was selected for the elution system. When using methanol and acetonitrile as organic phases to elute the ellagic acid reference standard, it was found that the retention time of ellagic acid was shorter in the acetonitrile elution system, and fewer impurity peaks appeared before and after the target peak in the sample when the volume ratio of water-acetonitrile was 85:15. This facilitated qualitative and quantitative analysis and significantly improved detection efficiency.
Therefore, the mobile phase for the quantitative determination of ellagic acid in Pu'er ripened tea was determined to be 0.2% phosphoric acid solution-acetonitrile (volume ratio 85:15), with a retention time of 16.0 min (±0.5 min), as shown in Figure 2.
2. Optimization of Sample Pretreatment
The effects of different material-to-solvent ratios, different types of extraction solvents, different extraction times, and different numbers of extractions on the extraction efficiency of ellagic acid