Chemicals and equipment
Gallic acid, 2-deoxy-D-ribose, Folin-Ciocalteu’s reagent and other chemicals needed for cell culture and cell viability studies were purchased from Sigma Chemicals Co. (P.O. Box 14508, St. Louis, MO 63178 USA). 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical, epigallocatechin gallate (EGCG), aluminium chloride, polyvinyl-polypyrrolidone (PVPP) were purchased from Fluka (Flukachemie GmbH, CH-9471 Buchs). L-Ascorbic acid, hydrogen peroxide, N-(1-naphthyl)-ethylene diaminedihydro chloride and ethanol were purchased from BDH Chemicals (BDH Chemicals Ltd, Poole, England). All chemicals used were of analytical grade.
SHIMADZU UV 1601 UV/Visible spectrophotometer (Shimadzu Corporation, Kyoto, Japan) was used to read the absorbance. LFT 600 EC freeze dryer was used to freeze-dry the plant extracts (LFT 600 EC, −90–95 °C temperature, 10 valves with Hitachi pump). Cells used for the assessment of anti-proliferative activity (RD, CC1) were incubated at 37 °C in a humidified CO2 incubator (SHEL LAB/Sheldon manufacturing Inc., Cornelius, OR 97113, USA). Olympus (1X70-S1F2) inverted fluorescence microscope (Olympus Optical Co. Ltd. Japan) and digital camera (MDC 200 (USB 2.0) 2 M pixels with CCD chip) were used for assessment and imaging of cell morphology.
RD cell line was kindly donated by Dr Sunethra Gunasena, Medical Research Institute, Colombo 08, Sri Lanka and CC1 (Normal rat liver fibroblast) cell line was obtained from Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi, Pakistan.
Plant materials
Phyllanthus debilis plants in the seeding stage were collected from Pannipitiya area in the Colombo district, Sri Lanka (June 2014). The plant was taxonomically identified and voucher specimens were deposited in Botany Department, Bandaranayaka Memorial Aurveda Research Institute, Nawinna, Colombo, Sri Lanka (Deposition number: 755/a).
Preparation of the decoctions
The plant materials were washed in distilled water followed by deionized water and air dried. The aerial parts and the root parts were separated and freeze dried until a constant weight was obtained. Dried samples were ground using a kitchen blender. The powdered aerial parts (50 g) were refluxed with 500 mL of deionized water for 3 h in triplicate. The roots were pooled together and the powdered roots (50 g) were refluxed as previously. Decoctions were filtered through a glass funnel plugged with cotton wool, then through a Whatman filter paper. Filtrate was centrifuged at 10,000 rpm for 15 min and the supernatant was freeze dried. The freeze dried samples were weighed and stored at −20 °C in sterile glass bottles until further use. The yield was calculated as a percentage of the dried plant material.
Removal of polyphenols
Polyphenols were removed using Polyvinyl polypyrrolidone (PVVP) column as described by Soysa (1997) [
12,
13]. Briefly, a cotton wool plug was placed inside a 5 cm
3 syringe after removing the plunger and the needle. Syringe was filled with PVPP (1.7 g). Water extract of AP and RP (3 mL) was layered over the PVPP column. The PVPP column was placed in a 15 mL falcon tube and centrifuged at 2,000 g for 10 min. Centrifugation was repeated for 6 times with the same column adding 1 mL of the extract and each fraction was collected to separate tubes. The first fraction was discarded and remaining fractions were analyzed for the presence polyphenols. The Absorbance of AP (aerial parts) and RP (root parts) before and after PVPP treatment were scanned for wavelengths using a UV/Visible scanning spectrophotometer.
Determination of total polyphenolic and flavonoid content
Total polyphenolic content (TPC) of the AP and RP were determined using Foiln-Ciocalteu’s method [
14]. Gallic acid was used to construct the standard curve and total phenolic content was expressed as w/w% gallic acid equivalents (GAE w/w %).
The flavonoid content of the extracts was determined by aluminum chloride colorimetric assay [
15]. Calibration curve was plotted using EGCG (− (−) -Epigallocatechingallate) standard and flavonoid content was expressed as w/w% EGCG equivalents (EGCGE w/w %).
Determination of total proanthocyanidins
A volume of 2 ml of vanillin reagent (1 g vanillin in 50 mL, 70 % H
2SO
4) was mixed with 200 μL of the sample (400, 500, 700 μg/mL). The resulting mixture was allowed to stand for 15 min at room temperature. The absorbance was determined at 500 nm [
16]. Calibration curve was constructed using EGCG and the total proanthocyanidin content was expressed as w/w % EGCG equivalent.
MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)) assay
Cells were plated in 24-well flat bottom tissue culture plates at a density of 2 × 10
5 cells/well and kept overnight at 37 °C in a CO
2 incubator to obtain a 70 % confluent mono layer. The cells were treated with AP, RP, PFAP and PFRP at different concentrations for 24 h. Cells for negative controls were exposed to the same conditions without the plant extract. Cycloheximide (0.1 %, 50 μl) served as the positive control. Medium was replaced by MEM (1 ml) and MTT reagent (100 μl, 5 mg/ml). The contents were incubated for 4 h at 37 °C. Isopropyl alcohol (750 μl) in 0.05 M HCl was added to dissolve MTT crystals. Absorbance was recorded at 570 nm. Percentage cell viability was calculated using the formula 2 [
21].
$$ \%\ Cytotoxicity = \frac{\left( Ab\ of\ Negative\ control- Ab\ of\ sample\right)\ X\ 100}{Ab\ of\ Negative\ control} $$
(2)
Statistics and calculations
Each experiment was performed at least in triplicate for three lyophilized samples prepared from aerial extracts and for the pooled sample (lyophilized) of the root extract. Calibration curves were considered as linear if R2 > 0.99. The EC50 values were calculated from either linear or logarithmic dose response curves where R2 > 0.95 was considered as linear. Student t test was carried out for the statistical calculations using Microsoft Excel.