1. Introduction
Oolong tea, a semi-fermented tea widely consumed in Asian countries, has been shown to contain an abundant amount of catechins, including epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC), epicatechin (EC), gallocatechin gallate (GCG) and catechin (C) [
1]. Among the various catechins in Oolong tea, EGCG was reported to be present in the largest amount while GCG was the lowest amount [
2]. Most importantly, catechins have been demonstrated to be protective against many types of chronic diseases such as inflammation, atherosclerosis and prostate cancer [
3].
Oolong tea beverage represents a vital product in the Taiwan beverage industry due to an increasing demand by consumers annually. Interestingly, Oolong tea leaf waste, a by-product produced during the processing of Oolong tea beverage, was also shown to contain a significant amount of catechins [
4]. Due to the great contribution of catechins to human health, it would be a great advantage to the food or drug industry if catechins can be further extracted from Oolong tea leaf waste and processed into functional food or even botanic drugs for possible clinical application in the treatment of chronic diseases. However, the high instability and poor bioavailability of catechins in vivo have limited their potential for further development into a botanic drug [
5]. Nevertheless, through preparation of nanoemulsions or microemulsions for encapsulation of catechins, both the catechin stability and bioavailability in vivo can be greatly enhanced to improve the anti-cancer effect substantially [
6].
Accordingly, both nanoemulsions and microemulsions are composed of oil, water, surfactant or co-surfactant in an appropriate proportion, with the former size ranging from 10 to 100 nm and the latter size from 2 to 100 nm [
7]. Furthermore, microemulsion is thermodynamically more stable than nanoemulsion, with a higher surface-to-mass ratio [
7]. The application of drug- or natural bioactive compound-loaded nanoemulsion or microemulsion in inhibiting cancer cell growth has been extensively studied. For example, Huang et al. [
8] prepared a lycopene–nanogold nanoemulsion with the average particle size being 21.3 nm by TEM analysis and demonstrated that it was effective in inhibiting the growth of colon cancer cell HT-29 through passive targeting by enhanced permeability and retention (EPR) effect. In other words, nanoemulsion is able to diffuse from the extracellular matrix into the cytoplasm and nucleus for antitumor efficiency [
8].
In the literature reports, most studies deal with the effects of catechin standards or green tea extracts on the growth of cancer cells. For instance, Carvalho et al. [
9] reported that the green tea extract was effective in inhibiting growth of kidney cancer cells A-497 and 769-P, with half the maximal inhibitory concentration (IC
50) being 54 μg/mL and 129 μg/mL, respectively. In a later study, Singh et al. [
10] further illustrated that EGCG, the most abundant catechin in green tea extract, was effective in inhibiting the growth of cervical cancer cells SiHa through an increase in both caspase-9 and caspase-3 activities. In a previous study, Tsai and Chen [
6] prepared a highly stable catechin nanoemulsion from green tea leaf waste and reported that it was effective in inhibiting the growth of prostate cancer cells PC-3 with the IC
50 being 8.5 μg/mL through activation of caspase-8, caspase-9 and caspase-3. However, the inhibition effect of catechin nanoemulsion prepared from Oolong tea leaf waste on the growth of prostate cancer cells DU-145 and mice tumors remains unexplored. The objective of this study was to prepare catechin nanoemulsion from Oolong tea leaf waste and demonstrate its efficiency in inhibiting the growth of prostate cancer cells DU-145 and mice tumors induced by DU-145.
3. Materials and Methods
3.1. Materials
A total of 12 kg of Oolong tea leaf waste (Camellia sinensis (L.) kuntze var.) were provided by a tea beverage processing company in Tainan, Taiwan. Prior to use, the Oolong tea leaf waste was freeze dried and then poured into 8 bags and vacuum sealed separately and stored in a −30 °C freezer. A total of 8 catechin standards including catechin, epicatechin, gallocatechin, epigallocatechin, gallocatechin gallate, epigallocatechin gallate, catechin gallate and epicatechin gallate, as well as internal standard L-tryptophan, were procured from Sigma-Aldrich Co. (St. Louis, MO, USA). The HPLC grade solvents methanol and acetonitrile were obtained from Merck Co. (Darmstadt, Germany). Both ethanol (99.9%) and formic acid as well as potassium dihydrogen phosphate were obtained from Sigma-Aldrich Co. Deionized water was madeusing a Milli-Q water purification system from Millipore Co. (Bedford, MA, USA). Lecithin was obtained from Cheng-Fung Co. (Taipei, Taiwan) while Tween 80 was acquired from Yi-Pa Co. (Taipei, Taiwan). The fixation solution (2.5% glutaraldehyde, 4% formaldehyde and 1% osmium tetroxide) and buffer solution (0.1 M Soreneon’s phosphate buffer) for TEM sample preparation were procured from Sigma-Aldrich Co., while the dehydration reagents (30%, 50%, 70%, 95% and 100% ethanol, and acetone), Spurr’s resin kit and EMS G300-Cu copper grid were obtained from Electron Microscopy Sciences (Hatfield, PA, USA).
Human prostate cancer cells (DU-145) and human fibroblast cells (CCD-986SK) with research resource identifier (RRID) of CVCL_0105 and CVCL_2400, respectively, were purchased from Bioresources Collection and Research Center, Taiwan Food Industry Development and Research Institute (Hsinchu, Taiwan). All experiments were performed with mycoplasma-free cells. The reagents for cell culture including sodium pyruvate, non-essential amino acid and penicillin-streptomycin were acquired from Gibco Co. (CA, USA), while minimum essential medium (MEM), Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS) and 0.25% trypsin-EDTA were from HyClone Co. (Longan, UT). Disodium hydrogen phosphate was procured from Panreac Quimica Co. (Needham Market, Suffolk, UK). Dimethyl sulfoxide (DMSO), hydrochloric acid, propidium iodide (PI), were obtained from Sigma-Aldrich Co. The MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent was obtained from USB Co. (Cleveland, OH, USA). Both caspase-3 assay kit and FITC Annexin V apoptosis detection kit were from BD Bioscience Co. (San Jose, CA, USA), while caspase-8 and caspase-9 fluorometric assay kits were from Bio Vision Co. (Milpitas, CA, USA).
For the animal study, a total of 36 4-week-old male BALB/c nude mice (specific pathogen free) were obtained from the National Experimental Animal Center (Taipei, Taiwan). Mouse EGF ELISA kit and VEGF ELISA kit were from Koma Biotech Co. (Seoul, Korea), while paclitaxel was obtained from Sigma-Aldrich Co.
3.2. Extraction of Catechins from Oolong Tea Leaf Waste
A method based on Tsai and Chen [
6] was modified to extract catechins from Oolong tea leaf waste. Briefly, a 0.2 g sample of Oolong tea leaf waste powder was mixed with 4 mL of 30%, 50% or 70% ethanol in water for comparison of extraction efficiency. Then, the mixture was sonicated in an ultrasonicator (DC400H, Hua-Hsia Co., Taipei, Taiwan) at room temperature for 1 h, followed by centrifuging in a high-speed centrifuge (Sorvall RC6 plus, Thermo Fisher Scientific Co., San Jose, CA, USA) at 4000 rpm (25 °C) for 30 min and collecting the supernatant, which was then filtered through a 0.6 μm glass filter paper, evaporated to dryness under vacuum, dissolved in 5 mL of 50% ethanol in water, filtered through a 0.22 μm membrane filter and stored at −20 °C for use.
3.3. HPLC Separation of Catechins
The separation condition of catechins in Oolong tea leaf waste was based on a report by Tsai and Chen [
6] and modified. An HPLC system from Agilent (model 1200 series, Agilent Technologies Co., Santa Clara, CA, USA) coupled with a single quadrupole mass spectrometer (model 6130) and a Gemini C18 column (250 × 4.6 mm ID, 5 μm particle size) from Phenomenex Co. (Torrance, CA, USA) was used with flow rate at 1 mL/min, column temperature at 30 °C, detection wavelengths at 245 nm and 280 nm and a gradient solvent system of 0.1% formic acid solution (A) and acetonitrile (B) was used: 88% A and 12% B initially, maintained for 3 min, then changed to 80% A and 20% B in 9 min, 75% A and 25% B in 12 min, maintained for 13 min, and returned to the original ratio. A total of five catechins in Oolong tea leaf waste were separated within 17 min.
3.4. Identification of Catechins
For identification of the various catechins in Oolong tea leaf waste, both retention time and mass spectra of unknown peaks in the HPLC chromatogram were compared with authentic standards and those reported in the literature. The peak purity was automatically determined by a photodiode array detector. A single quadrupole mass spectrometer with ESI mode (negative) was used for identification with the same parameters as reported by Tsai and Chen [
6]. For positive identification, an HPLC-MS/MS instrument with ESI mode (LTQ Orbitrap XL, Thermo Fisher Scientific Co., San Jose, CA, USA) was used in the scanning range from 100–1000 m/z with heated temperature at 300 °C, sheath gas flow rate at 50 arbitrary units, aux gas flow rate at 25 arbitrary units, sweep gas flow rate at 0 arbitrary units, spray voltage at 3 kV, capillary temperature at 275 °C, capillary voltage at −35 V and tube lens voltage at −100 V.
3.5. Validation of Quality Control Parameters
Both intra-day and inter-day variabilities were determined for method validation [
29], with the former being conducted by collecting catechin sample solution containing the internal standard L-tryptophan (10 μg/mL) for analysis in the morning, afternoon and evening on the same day in triplicate for a total of 9 analyses, while the latter was carried out by collecting catechin sample solution containing L-tryptophan (10 μg/mL) for analysis in the morning, afternoon and evening on the first, second and third day in triplicate, separately, for a total of 27 analyses.
For LOD and LOQ determination, three concentrations (0.2, 0.4 and 0.6 μg/mL) in 50% ethanol were prepared for four catechin standards including EGC, EC, EGCG and GCG separately. For ECG, 0.2, 0.25 and 0.3 μg/mL were prepared. Each concentration was injected into HPLC three times and the standard curves were prepared by plotting concentration against average peak area; from the slope (S) and maximum noise height (N), both LOD and LOQ were calculated based on S/N ≥ 3 and S/N ≥ 10, respectively [
6].
The recovery was determined by adding two levels of catechin standards including EGC (700 and 1050 μg), EC (600 and 900 μg), EGCG (3000 and 4500 μg), GCG (150 and 225 μg) and ECG (1000 and 1500 μg) to 0.2 g of Oolong tea leaf waste samples for extraction and HPLC analysis. The recovery was obtained based on the relative ratio of catechin level after HPLC to that before HPLC.
3.6. Quantitation of Catechins
For quantitation, a total of seven concentrations were prepared for EGC (85, 100, 115, 130, 145, 160 and 175 μg/mL), EC (40, 45, 50, 55, 60, 65 and 70 μg/mL), EGCG (355, 370, 385, 400, 415, 430 and 445 μg/mL), GCG (5, 10, 15, 20, 25, 30 and 35 μg/mL) and ECG (85, 100, 115, 130, 145, 160 and 175 μg/mL). Then, each concentration was mixed with L-tryptophan at a fixed concentration at 10 μg/mL. After injection into HPLC three times for each concentration, the standard calibration curves were prepared by plotting the concentration ratio (standard versus IS) against the area ratio (standard versus IS), and both the linear regression equations and coefficient of determination (R
2) were automatically obtained by a Microsoft EXCEL software system. The contents of each catechin (μg/g) were then quantified [
6].
3.7. Preparation of Catechin Nanoemulsion
A sample of catechin extract (0.892 mL) containing catechin at 11,208.2 μg/mL was poured into a tube and evaporated to dryness under nitrogen. Then, 0.05 g of lecithin in liquid form (0.5%) and 0.7 g of Tween 80 (7%) were added sequentially with thorough stirring after each addition, followed by adding 9.25 g deionized water (92.5%), stirring thoroughly and sonicating the solution for 1.5 h to obtain a transparent catechin nanoemulsion of 10 mL with a yellow appearance containing total catechins at 1000 μg/mL.
3.8. Characterization of Catechin Nanoemulsion
A portion of catechin nanoemulsion (100 μL) was collected and diluted 30 times with 25 mM of potassium dihydrogen phosphate buffer solution (pH 5.3–5.5), after which the solution was poured into a polystyrene tube and the particle size distribution and mean particle size were determined by a dynamic light scattering instrument (DLS) from Brookhaven Instruments Co. (Holtsville, NY, USA) with a BIC Particle Sizing 90 Plus software system. A portion of catechin nanoemulsion (100 μL) was diluted with deionized water 30 times, after which 300 μL were collected and the zeta potential was determined in a SZ-100 model zeta potential analyzer from Horiba Scientific Co. (Kyoto, Japan). A portion of catechin nanoemulsion was diluted with deionized water 100 times, after which 20 μL were collected and dropped onto a copper grid for 45 s and the excessive sample was removed with a glass filter paper. Then, 2% (20 μL) phosphotungstic acid (PTA) was added for negative staining for 30 s, and the excessive PTA was removed with a glass filter paper and sample placed in a moisture-proof box for drying. Both the size and shape of catechin nanoemulsions were determined by a JEM-1400 model TEM (JEOL Co., Tokyo, Japan) by enlarging the sample solution 3 × 10
5 times and observing it under 120 kV. In addition, a portion (100 μL) of catechin nanoemulsion was collected and diluted 10 times with 25 mM potassium dihydrogen phosphate buffer solution (pH 5.3–5.5). Then, the solution was poured into a centrifuge tube containing 3 kDa of dialysis membrane for centrifugation in a DSC-301SD LED model microcentrifuge (Digisystem Laboratory Instruments Co., New Taipei City, Taiwan) at 12,000 rpm (25 °C) for 20 min. Unencapsulated (free) catechin could penetrate into the dialysis membrane, and the lower layer (200 μL) was collected and evaporated to dryness under nitrogen, and 100 μL internal standard (10 μg/mL) was added for HPLC analysis. The encapsulation efficiency was calculated using the following formula:
For the stability study, catechin nanoemulsion was stored at 4 °C for 90 days, during which a portion of the sample was collected every 15 days for the determination of particle size distribution by DLS and zeta potential. Also, a portion of catechin nanoemulsion (200 μL) was poured into a tube and placed into a water bath with temperatures controlled at 40, 50, 60, 70, 80, 90 and 100 °C separately and heated for 0.5, 1, 1.5 and 2 h. Both particle size distribution and zeta potential were also analyzed.
3.9. Cell Culture for Prostate Cancer Cells DU-145
Prostate cancer cells DU-145 were cultured in MEM medium by mixing 10.1 g MEM powder with 700 mL deionized water, 2.2 g sodium bicarbonate and 80 mL FBS, 10 mL penicillin-streptomycin, and diluted to 1 L with deionized water (pH 7.2–7.4). Similarly, human fibroblast cells CCD-986SK were cultured in DMEM medium, prepared by mixing 700 mL deionized water with 1 L medium powder, 1.5 g sodium bicarbonate, 100 mL FBS, 10 mL penicillin-streptomycin and 10 mL sodium pyruvate (100 mM), and diluted to 1 L with deionized water (pH 7.2–7.4). Both media were filtered through a 0.2 μm membrane filter and stored at 4 °C for use. For subculture of DU-145 or CCD-986SK cells, the medium in a culture plate was removed, washed with PBS and one mL of 0.25% trypsin-EDTA was added for incubation (SCA-165DS CO2 incubator, Astec Co., Fukuoka, Japan) for 3–5 min. Then, 1 mL of medium was added to terminate trypsin-EDTA reaction, after which the medium was centrifuged at 1500 rpm (25 °C) for 5 min and the supernatant was aspirated. Then, 1 mL of MEM or DMEM was added, followed by collecting an appropriate amount of cells for seeding in a culture plate containing fresh medium.
3.10. MTT Assay for DU-145 and CCD-986SK Cells
A 50 mg MTT powder was dissolved in 10 mL of sterilized PBS and filtered through a 0.22 μm membrane filter to obtain a stock solution of 5 mg/mL. Prior to use, the MTT stock solution was mixed with Hank’s balanced salt solution (HBSS) at 1:9 (
v/
v). Cells were seeded in a 96-well plate with each well containing 1 × 10
4 cells. After incubation overnight for cell attachment, the medium was removed and replaced with various concentrations of catechin extract or catechin nanoemulsion for 48 h incubation. Then, the medium was discarded and 200 μL of MTT (0.5 mg/mL in PBS) were added and cultured for 2 h, after which the MTT solution was removed, 100 μL of DMSO was added and stirred for 10 min and the absorbance was measured at 570 nm with an ELISA reader (Molecular Devices Co., Sunnyvale, CA, USA). The relative cell survival rate was calculated using a formula as described by Huang, Wei, Inbaraj and Chen [
8].
3.11. Cell Cycle Analysis for DU-145 Cells
Cells were seeded in a 6-well plate with each well containing 1 × 106 cells. After incubation overnight for cell attachment, the medium was removed and replaced with various concentrations of catechin extract or nanoemulsion for 48 h incubation. Then, the medium was transferred to a centrifuge tube, washed with PBS and 0.5 mL of trypsin-EDTA (0.25%) was added for a 10 min reaction. After centrifugation at 1500 rpm for 5 min (4 °C), the supernatant was removed and the medium washed with PBS (0.5 mL) twice. Next, one mL of 70% ethanol (4 °C) was added for cell fixation at 4 °C, centrifuged at 1500 rpm for 5 min (4 °C) and the supernatant was removed, followed by washing with 0.5 mL of PBS twice, adding 0.8 mL of PBS, 0.1 mL of RNase (1 mg/mL) and 0.1 mL of propidium iodide (100 μg/mL) for reaction in a water bath for 30 min in the dark. After filtration through a 40 μm nylon filter, cells were analyzed for the proportions of sub-G1, G0/G1, S and G2/M phases by a flow cytometer (Beckman Coulter Co., Tokyo, Japan) with a Partec Flow Maz version 2.4d software system.
3.12. Annexin V/propidium Iodide Staining Assay for DU-145 Cells
Cells were seeded in a 6-well plate with each well containing 1 × 106 cells. After incubation overnight for cell attachment, the medium was removed and replaced with various concentrations of catechin extract or nanoemulsion for 48 h incubation. Then, the medium was transferred to a centrifuge tube, washed with PBS, and 0.5 mL of trypsin-EDTA (0.25%) added for 10 min reaction. After centrifugation at 1500 rpm for 5 min (4 °C), the supernatant was removed and the medium washed with PBS twice. Then, 0.1 mL of binding buffer was added to suspend cells, followed by adding 5 μL of Annexin V-FITC and 10 μL of PI for 15 min reaction in the dark and subsequent analysis of apoptotic and necrotic cell populations by a flow cytometer with a Partec Flow Maz version 2.4d software system.
3.13. Activities of Caspase-3, Caspase-8 and Caspase-9
The activity of caspase-3 was determined by a fluorescence assay kit. Briefly, a sample of DU-145 cell lysate (25 μL) containing 40 μg cell protein was mixed with 100 μL of 1XHEPES buffer, after which the solution was reacted in a 37 °C water bath for 1 h in the dark and then transferred to a 96-well plate for absorbance measurement at 380 nm (excitation wavelength) and 440 nm (emission wavelength) by using a multimode microplate reader. Similarly, both caspase-8 and caspase-9 activities were determined by collecting 50 μL of DU-145 cell lysate separately and mixing with 50 μL of 2X reaction buffer, reacted in a 37 °C water bath for 1 h in the dark and then transferred to a 96-well plate for absorbance measurement at 400 nm (excitation wavelength) and 505 nm (emission wavelength) by using a multimode microplate reader (Infinite 200PRO, Tecan Co., Mannedorf, Switzerland).
3.14. Animal Study Approval and Handling for Evaluating Anti-Tumor Efficiency in Mice
A total of 36 4-week-old SPF (specific pathogen free) male BALB/c nude mice were obtained from the National Animal Research Center (Taipei, Taiwan) and transported to Fu Jen University Animal Research Center. A prior approval of animal experiment protocols was obtained from Fu Jen Catholic University Animal Subjects Review Committee and strict regulations on human care for laboratory animals was adopted based on approved guidelines [
30]. Each mouse was raised in an individual ventilated cage and fed with LabDiet 5010 with temperature at 21 ± 2 °C and relative humidity at 55 ± 10% for 12 h under light and 12 h in the dark. All the procedures followed the standard operation method for experimental animals.
3.15. Animal Experiments
After an adaptation period of 2 weeks, about 1 × 10
7 prostate cancer cells DU-145 were injected into flank of each mice for 28 days to induce tumor formation, then the tumor size was measured with a caliper every 3 days. After the average tumor volume reached about 200 mm
3, we started the injection of catechin extract and catechin nanoemulsion. Both tumor size and mice weight were recorded every 3 days. Tumor volume was calculated using the following formula:
where V, L and W denote tumor volume, length and width, respectively. Then, 36 male BALB/c nude mice were divided into 6 groups with 6 mice each and with intraperitoneal injection (IP) every 3 days for a total of 6 injections: (1) control group—injection with 0.2 mL of saline solution (0.85%), (2) drug group—injection with 0.2 mL of paclitaxel in deionized water (10 mg/kg BW), (3) low dose (10 mg/kg BW) of catechin extract group—injection with 0.2 mL of catechin extract (evaporated to dryness and dissolved in deionized water), (4) high dose (20 mg/kg BW) of catechin extract group—injection with 0.2 mL of catechin extract (evaporated to dryness and dissolved in deionized water), (5) low dose (10 mg/kg BW) of catechin nanoemulsion group—injection with 0.2 mL of catechin nanoemulsion and (6) high dose (20 mg/kg BW) of catechin nanoemulsion group—injection with 0.2 mL of catechin nanoemulsion. Then, all the mice were placed under euthanasia with CO
2 and the tumors were collected for weight measurement. The heart blood was collected and poured into a tube for centrifugation at 3500 rpm (4 °C) for 20 min, and serum in the upper layer was collected in a tube for storage at −80 °C.
3.16. Determination of Growth Factors in Serum
Both levels of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) in serum were measured with an ELISA kit. In brief, a 96-well plate containing antibody was washed with 300 μL of wash buffer (0.05% Tween 20 in PBS) 4 times, and a volume (100 μL) of EGF or VEGF standard with various concentrations (15.625, 31.25, 62.5, 125, 250, 500 and 1000 pg/mL) or serum was added to the 96-well plate for a 2 h reaction at room temperature. Then, the plate was washed with 300 μL of wash buffer 4 times, followed by adding 100 μL of detection antibody for a 2 h reaction and then 100 μL of streptavidin-HRP conjugate for a 30 min reaction. The plate was washed again with 300 μL of wash buffer 4 times, after which 100 μL of the solution containing tetramethyl benzidine (TMB) was added for 5 min reaction and then 100 μL of stop buffer (2M H2SO4) was added to terminate the reaction. The absorbance was measured at 450 nm and both levels of EGF and VEGF were obtained based on the linear regression equations of the standard curves.
3.17. Statistical Analysis
All the data were analyzed using the statistical analysis system and subjected to ANOVA analysis and Duncan’s multiple range test for significance in mean comparison (p < 0.05).