Plant materials
Dried flowers of C. morifolium Ramat. originated from Hang Zhou, China was procured from a Chinese drug store (Lee Mun Onn Medicine Store) in Petaling Jaya (GPS coordinates: 3.0875° N, 101.6451° E), Selangor, Malaysia in May 2017. Authentication of the plant sample was carried out by a botanist, Dr. Mohd. Firdaus Ismail at the Biodiversity Unit, Institute of Bioscience, Universiti Putra Malaysia, and a voucher specimen (SK3139/17) was deposited at the unit’s herbarium of that institute.
Chemicals and reagents
HPLC grade solvents such as methanol, petroleum ether, ethyl acetate, dimethyl sulfoxide (DMSO), hexane, chloroform, and acetonitrile as well as celite and culture Dulbecco’s Modified Eagle Medium: Nutrient Mixture F–12 (DMEM-F12) were purchased from ThermoFisher Scientific (Waltham, United States). Potassium phosphates monobasic, dibasic, xanthine, allopurinol, crystal violet, trypan blue staining solution, sodium dodecyl sulfate (SDS), 2’–7’dichlorofluorescin diacetate (DCFH-DA), tert-butyl hydroperoxide (tBuOOH), 5-sulfosalicyclic acid dihydrate (SSA), and 2-Vinylpyridine were purchased from Sigma Aldrich (St. Louis, United States). Enzyme of xanthine oxidase from cow’s milk was purchased from Roche Diagnostics (Mannheim, Germany). Preparative layer plates (Silica gel 60 F254, 20 cm × 20 cm) and silica gel 60 (230–400 meshes ASTM; 0.040–0.063mm)was purchased from Merck (New Jersey, United States). Phosphate buffered saline was purchased from First Base Laboratories Sdn Bhd (Selangor, Malaysia). SYBR Green Master Mix was purchased from Bio-Rad Laboratories (California, United States) and ultrapure water from a Mili-Q® purification system was used in this research.
Plant extraction and solvent-solvent partition
Dried flowers of C. morifolium were powdered and approximately 500 g of the dried powder was sonicated in 80% methanol (2 L) for 2 hours at 25°C. The 80% hydromethanolic filtrate was evaporated using rotary evaporator to obtain a crude extract. The crude extract (58.86 g) was dissolved in 400 mL of methanol and then partitioned using equal volume of petroleum ether and ethyl acetate. Each fraction was then concentrated under reduced pressure to obtain petroleum ether (PE) fraction, ethyl acetate (EtOAc) fraction and residual (RS) fraction. All fractions were weighed and stored at –80oC until further use.
In vitro xanthine oxidase inhibitory activity
The XO inhibitory activity was determined based on a modified method [9]. The substrate and the enzyme solutions were prepared immediately before use. Test samples were dissolved in DMSO and diluted to different concentrations (12.5 - 400 µg/mL). The assay mixture consisted of 130 µL of 0.05 M potassium phosphate buffer (pH 7.5), 10 µL of sample, 10 µL of 0.1 unit/mL XO enzyme solution. After pre-incubation for 10 minutes at 25°C, the reaction was then initiated by the addition of 100 µL xanthine (0.15 mM) solution and incubated for 10 minutes at 37°C. The enzymatic conversion of xanthine to form uric acid was measured at absorbance of 295nm. All reactions were performed in triplicates (n = 3) in a 96-well UV microplate. Negative control containing the assay mixture without sample extract was also prepared. Allopurinol was used as a positive control in the assay mixture. The IC50 for allopurinol and all the fractions were determined. The XO inhibitory activity was calculated as:
XO Inhibition (%) = (Control absorbance—Sample absorbance) / Control absorbance x 100%
Xanthine oxidase enzyme kinetic
In order to determine the inhibitory mode of the active fractions and sub-fractions, Lineweaver-Burk plot analysis was performed. The kinetic study was carried out in the absence and presence of inhibitor with varying concentrations (0.075—2.4 mM) of xanthine as substrate, by using the XO inhibitory activity assay. The mode of inhibition was determined from the slope of the Lineweaver-Burk plot for competitive, non-competitive and mixed-type inhibition.
Bioassay-guided fractionation using column chromatography
Approximately 2.50 g of the most bioactive fraction of C. morifolium was subjected to silica gel open column chromatography using silica gel 60 (230—400 meshes ASTM; 0.040—0.063mm; Merck) as stationary phase. Separation was accomplished by eluting various solvent mixtures using mobile phase of increasing polarity from 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, 100:0 (v/v) of hexane: ethyl acetate. The residuals in the column were recovered by sequential elution of equal volume of methanol, then followed by equal volume of methanol: chloroform (1:1, v/v). The sub-fractions were collected and evaporated. They were then subjected to thin layer chromatography (TLC) (Silica gel 60 F254 20cm × 20cm; Merck) profiling by using hexane: ethyl acetate (1:1) as mobile phase. The sub-fractions with the same retention factor (Rf) were pooled together to afford 15 sub-fractions (F1—F15). All sub-fractions were subjected to XO inhibitory assay and the bioactive sub-fractions were fractionated further. Refer to Figure 1 in results for flow chart of bioassay-guided fractionation.
Cellular assays
Crystal violet assay
Crystal violet assay was performed based on a modified protocol [10]. HepG2 cell line was used in this assay. The cells were seeded in a 96-well plate and incubated for 24 hours at 37oC in CO2 incubator. Medium was aspirated from wells, washed twice with 1× phosphate-buffered saline (PBS) solution and 100 µL/well of fresh medium containing sub-fraction (0.1—100 µg/mL) was added into the wells. Cells were incubated for 24 hours at 37oC in CO2 incubator with the treatments condition. After 24 hours incubation, medium was aspirated and the cells were washed twice with 1× PBS solution. Next, 50 µL of 0.5% crystal violet staining solution was added into each well and incubated for 20 minutes in CO2 incubator at 37oC. Crystal violet staining solution was removed and the plate was washed twice with milliQ water. Next, 1% of sodium dodecyl sulfate (SDS) solution was added into each well of plate and incubated for 20 minutes. The optical density of each well at 570nm (OD570) was measured with 96-well UV microplate reader (Infinite M200Pro, Tecan Group Ltd, Männedorf, Switzerland). The percentage of viable cells were determined by comparing the average OD570 values of extract treated cells with the OD570 values of the non-treated cells. The means and the standard error of mean are calculated for at least three independent experiments.
Trypan blue exclusion assay
HepG2 cells were seeded and grown in a 6-well plate. Medium was washed and replaced until the cells confluency reached 70%. Cells were treated with different sub-fractions and incubated for 24 hours in CO2 incubator at 37oC. Medium was aspirated and 0.5mL trypsin was added and incubated for 5 minutes. Next, cell suspension was collected into tube accordingly. Tubes containing HepG2 cells were re-suspended. Cells were then stained by combining 20 µL of cell sample with 20 µL of trypan blue staining solution to obtain 0.1% final concentration. Then, 20 µL of mixed cell sample was loaded into the Nexcelom Cellometer counting chamber and inserted into Auto T4 sample slot. Cells sample was analysed. Live cells as well as dead cells, total cells, percentage of viability, live cell concentration and total cell concentration were determined and calculated [11, 12].
Reactive Oxygen Species (ROS) assay
HepG2 cells were seeded and grown in a 24-well plate. Medium was washed and replaced until the cells confluency reached 70%. Medium was changed to DMEM-F12 without fetal bovine serum and incubated for 24 hours in CO2 incubator at 37oC. Then, 10 µL of 2’, 7’-Dichlorodihydrofluorescein diacetate (5 µM) reagent was added into wells and incubated in CO2 incubator for 45 minutes. Medium was washed out using 1 mL of 1× PBS solution twice. Cells were treated with 500 µL of tert-butyl hydroperoxide (400 µM) as positive control and test samples at different concentrations (0.1—10 µg/mL). Absorbance was obtained immediately at excitation of 485 nm/± 20 and emission of 528nm/± 20 and repeated every 30 minutes for 2 hours [13].
In vivo anti-hyperuricemic effects of C. morifolium in potassium oxonate-induced rats
Male SD rats (7 weeks) were acclimatized to the laboratory condition on normal diet for at least one week prior to the experiment. Animals were provided with rodent diet and clean water ad libitum, except 1 hour prior to drug administration, whereby access to food was restricted. All the rats were randomly divided into five groups of six (n = 6) rats each. Experimental hyperuricemia was induced by injection of uricase inhibitor, potassium oxonate (PO). The rats were intraperitoneally (i.p.) injected with PBS containing 250 mg/kg PO (except normal control group) one hour before the administration of test samples to increase the serum uric acid (SUA) level on daily basis, for 7 days. After one hour, the rats were treated with 25 mg/kg and 50 mg/kg of C. morifolium EtOAc F10 via oral gavage. The extract was replaced by PBS (pH 7.4) and water in negative and normal control groups, respectively.
Allopurinol (10 mg/kg) was used as positive control in this study. Blood samples were collected by rats’ tail vein bleeding on day 7. On day 6, each rat was kept separately for urine collection using single animal method [14]. On day 8, the rats were euthanized under CO2 and blood samples were collected by cardiac puncture. Blood samples were transferred to sterile tubes and allowed to clot on ice for 30 minutes. The blood was centrifuged at 2,000g for 15 minutes at 4oC to obtain serum. The urine and serum samples were stored at –80°C until assays. Livers were also dissected immediately on ice and homogenized at frequency of 20 kHz for 5 minutes. The homogenate was freeze in liquid nitrogen and stored at –80°C until XO activity and gene expression analysis. SUA and urine uric acid (UUA) levels were measured using Amplex® Red uric acid/uricase assay kit (ThermoFisher Scientific, United States) while liver XO activity was measured by using Amplex® Red xanthine/xanthine oxidase assay kit (ThermoFisher Scientific, United States).
qPCR analysis of XO gene expression
qPCR analysis was carried out using Bio-Rad CFX96Real-Time PCR, version 3.1to determine the expression level of XO after drug administration. Three rats that had serum uric acid (SUA) levels close to the mean value in each group on day 7 were selected. Total RNA was extracted from their livers using NucleoSpin RNA kit (Macherey-Nagel GmbH & Co. KG, Germany). Next, RNA quality and quantity were determined by performing agarose gel electrophoresis and Nanodrop spectrophotometry. One-step Real-Time SYBR Green qPCR was performed. The primers (forward primer: 5’-GCATGCCAGACCATACTGAA–3’; and the reverse primer: 5’-AAATCCAGTTGCGGACAAAC–3’) for rat xanthine dehydrogenase (XDH; Gene ID: 497811, mRNA sequence: NM_017154) were purchased from IDT Singapore Pte Ltd, Singapore. The housekeeping gene, β-actin for rat (mRNA sequence: NM_031144) with primer sequences of 5’-ATTGTGATGGACTCCGGAGA–3’ (forward) and 5’-CAGCTCATAGCTCTTCTCCA–3’ (reverse) was purchased from MYTACG Bioscience Enterprise, Malaysia.
Putative identification of XO inhibitors using HPLC-Q-TOF-MS/MS
EtOAc sub-fractions were analysed by using Hypersil GOLD C18 column (150 × 4.6mm, 5µm, ThermoFisher Scienctific, United States) at 25°C on Agilent 1260 Infinity HPLC with UV detector at 210 nm, 254 nm, and 280 nm wavelengths.The mobile phase consisted of water (A) and acetonitrile (B) with gradient elution (0—5 minutes, 98% A, 0.7 mL/min; 5—7 minutes, 85% A, 0.5 mL/min; 7—20 minutes, 25% A, 0.2 mL/min; 20—40 minutes, 15% A, 0.2 mL/min; 40—45 minutes, 10% A, 0.5 mL/min; 45—50 minutes, 0% A, 0.7 mL/min; 50—55 minutes, 98% A, 0.7 mL/min.). Test sample of 20 µL was injected in each run. Samples were prepared at 10 ppm in HPLC grade methanol, filtered through 0.45 µm syringe filter (ThermoFisher Scientific, United States) and sent to Infra Advanced Laboratory, University of Malaya for the identification of compounds by using Agilent 6200 series TOF/6500 series Q-TOF (Santa Clara, United States) with ESI source (Santa Clara, United States). HPLC-Q-TOF-MS/MS was performed in positive mode under the following operating parameters: scanning mode, total ion current (TIC) and extracted ion chromatogram (EIC); scanning range, 10—1700 m/z; fragmentor voltage, 175V; Gas flow, 14 L/minutes; and nebulizer, 35 psig. The mass spectrometry data were qualitatively analysed using MassHunter Workstation software (version B.05.01, Agilent Technologies) for possible markers and were compared to standard online database such as METLIN metabolite database, Human Metabolome Database (HMDB), PubChem and KEGG metabolite databases.
Statistical analysis
All data were analysed using Statistical Package for Social Sciences (SPSS) version 21.0 and expressed as mean ± standard error of mean (SEM). The data in the present study was subjected to One-way analysis of variance (ANOVA) and the significance of difference between the means was determined by post-hoc Duncan’s multiple range tests at 95% least significant difference (p ≤ 0.05).