Preparation of catechin extracts and nanoemulsions from green tea leaf waste and their inhibition effect on prostate cancer cell PC-3

Abstract

Green tea is one of the most commonly consumed natural health beverages in Taiwan's market, with the major functional component catechin being shown to possess several biological activities such as antioxidation, anticancer, and prevention of cardiovascular disease. The objectives of this study were to develop a high-performance liquid chromatography-mass spectrometry method to determine the variety and content of catechins in green tea leaf waste, a by-product obtained during processing of tea beverage. In addition, catechin nanoemulsion was prepared to study its inhibition effect on prostate cancer cell PC-3. Results showed that a total of eight catechin standards were separated within 25 minutes by using a Gemini C18 column and a gradient mobile phase of 0.1% formic acid (A) and acetonitrile (B) with flow rate at 1 mL/min, column temperature at 30°C, and detection wavelength at 280 nm. Among various extraction solvents, 50% ethanol generated the highest yield of total catechins from tea leaf waste, of which five catechins were identified and quantified. The catechin nanoemulsion was composed of catechin extract, lecithin, Tween 80, and deionized water in an appropriate proportion, with the mean particle size being 11.45 nm, encapsulation efficiency 88.1%, and zeta potential -66.3 mV. A high stability of catechin nanoemulsion was shown over a storage period of 120 days at 4°C. Both catechin extract and nanoemulsion could inhibit growth of PC-3 tumor cells, with the half maximal inhibitory concentration being 15.4 μg/mL and 8.5 μg/mL, respectively. The PC-3 cell cycle was arrested at S phase through elevation of P27 expression and decline of cyclin A, cyclin B, cyclin-dependent kinase 2, and cyclin-dependent kinase 1 expression. In addition, both catechin extract and nanoemulsion could induce apoptosis of PC-3 cells through decrease in B-cell lymphoma 2 (bcl-2) expression and increase in cytochrome c expression for activation of caspase-3, caspase-8, and caspase-9. Taken together, both caspase-dependent and caspase-independent pathways may be involved in apoptosis of PC-3 cells.

Keywords: HPLC-MS; apoptosis; catechin nanoemulsion; green tea leaf waste; prostate cancer cell PC-3.

Figure 1

Effect of different ethanol proportions on the catechin contents in green tea leaf waste extracts. Notes: Results are presented as mean ± standard deviation of triplicate determinations. Data with different capital letters (A–C) on each bar represent the content of each catechin or total catechin extracted using different solvents are significantly different at P<0.05. Abbreviations: GC, gallocatechin; EGC, epigallocatechin; EGCG, epigallocatechin gallate; GCG, gallocatechin gallate; ECG, epicatechin gallate.

Figure 2

High-performance liquid chromatograms of catechin standards detected at 280 nm (A) and green tea leaf waste extract at 280 nm (B) as well as 245 nm (C). Notes: Peaks: 1, GC; 2, EGC; 3, C; 4, EC; 5, EGCG; 6, GCG; 7, ECG; 8, CG; internal standard (l-tryptophan). Abbreviations: GC, gallocatechin; EGC, epigallocatechin; C, catechin; EC, epicatechin; EGCG, epigallocatechin gallate; GCG, gallocatechin gallate; ECG, epicatechin gallate; CG, catechin gallate; IS, internal standard; min, minutes.

Figure 3

Particle size distribution of blank nanoemulsion (A) as well as catechin nanoemulsion with filtration (B) and without filtration (C) along with TEM images of catechin nanoemulsion captured at two different magnifications (D and E). Abbreviation: TEM, transmission electron microscope.

Figure 4

Effects of different levels of 50% ethanol (A) and blank nanoemulsion (B) on the growth of both CCD-986SK fibroblast cells and PC-3 prostate cancer cells as well as the effects of catechin extract and nanoemulsion on CCD-986SK cells (C and D) and PC-3 cells (E and F). Notes: Results are presented as mean ± standard deviation of triplicate analyses. Data with different capital letters (A–E) on each bar in (A), (B), (E), and (F) represent the PC-3 cell viability at different concentrations of ethanol, blank nanoemulsion, catechin extract, and catechin nanoemulsion and are significantly different at P<0.05 compared to the control, respectively. Data with different small letters (a–f) on each bar in (A), (B), (C), and (D) represent the CCD-986SK cell viability at different concentrations of ethanol, blank nanoemulsion, catechin extract, and catechin nanoemulsion and are significantly different at P<0.05 compared to the control, respectively.

Figure 5

Effects of catechin extract and nanoemulsion on P27 (A), CDK2 (B), CDK1 (C), cyclin A (D), cyclin B (E), bcl-2 (F), and cytochrome C (G) protein expressions in PC-3 prostate cancer cells. Notes: Results are presented as mean ± standard deviation of triplicate analyses. Data with different capital letters (A–E) on each bar represent the ratio of each protein expression relative to GAPDH at different concentrations of catechin extract or nanoemulsion and are significantly different at P<0.05 compared to the control. The abbreviations used in X-axes indicate control (C), catechin extract (E), and catechin nanoemulsion (N). Abbreviations: CDK, cyclin-dependent kinase; bcl-2, B-cell lymphoma 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 5

Effects of catechin extract and nanoemulsion on P27 (A), CDK2 (B), CDK1 (C), cyclin A (D), cyclin B (E), bcl-2 (F), and cytochrome C (G) protein expressions in PC-3 prostate cancer cells. Notes: Results are presented as mean ± standard deviation of triplicate analyses. Data with different capital letters (A–E) on each bar represent the ratio of each protein expression relative to GAPDH at different concentrations of catechin extract or nanoemulsion and are significantly different at P<0.05 compared to the control. The abbreviations used in X-axes indicate control (C), catechin extract (E), and catechin nanoemulsion (N). Abbreviations: CDK, cyclin-dependent kinase; bcl-2, B-cell lymphoma 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 6

Activities of caspase-3 (A), caspase-8 (B), and caspase-9 (C) in PC-3 prostate cancer cells as affected by catechin extract and nanoemulsion from green tea leaf waste. Notes: Results are presented as mean ± standard deviation of triplicate analyses. Data with different capital letters (A–C) on each bar represent the caspase activity in PC-3 cells at different concentrations of catechin extract or nanoemulsion and are significantly different at P<0.05 compared to control. The abbreviations used in X-axes indicate control (C), catechin extract (E), and catechin nanoemulsion (N).