Kaempferol

DPPH (2,2-diphenyl-1-picrylhydrazyl); formic acid; vanillin; and standards of phenolic compounds, including phenolic acids such as gallic, caffeic, vanillic, salicylic, p-coumaric, and trans-ferulic acid; and flavonoids (hesperidin, quercetin, kaempferol, isorhamnetin, acacetin, hesperetin, pinocembrin, and chrysin) were purchased from Sigma-Aldrich (Poznań, Poland). Rutin trihydrate was purchased from Fluka (Poznań, Poland). Potassium hexacyanoferrate (II), K₄Fe(CN)₆ × 3H₂O; and zinc acetate, Zn(CH₃COO)₂ × 2H₂O, used for the preparation of Carrez reagents I and II, ethyl alcohol, Folin–Ciocâlteu reagent, and anhydrous sodium carbonate were purchased from POCH (Gliwice, Poland). J.T. Baker (Dventer, The Netherlands) supplied methanol for HPLC analyses and BakerBond C18-SPE 500 mg/6 mL solid-phase extraction columns. All reagents were of analytical-grade purity.

The cells of the PC12 adherent cell line (ATCC CRL-1721.1) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma, Aldrich, St. Louis, MO, USA), supplemented with 10% horse serum, 5% fetal bovine serum, and 1% antibiotic mixture containing penicillin–streptomycin, in a humidified atmosphere at 37 °C with 5% CO₂. The PC12 cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well and differentiated with 100 ng/mL nerve growth factor (NGF, Sigma-Aldrich, St. Louis, MO, USA) for 5 days into neuronal cells.
After the NGF-induced differentiation, the cells were exposed to 1 μg/mL lipopolysaccharide (LPS, Sigma-Aldrich) for 24 h to induce an inflammatory response. Subsequently, the cells were treated with different NCs (experimental groups) and vehicles (control), and the cells without LPS treatment were called the normal-control group. The NCs were various flavonoid compounds, including astragalin, dihydromyricetin, coumarin, quercetin, luteolin, chrysin, kaempferol, and apigenin (Sigma-Aldrich), at concentrations of 0, 1, 5, 25, and 25 μM. The flavonoid compounds were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) and diluted in a culture medium, with the final DMSO concentration not exceeding 0.1%. After 24 h of flavonoid treatment, cell viability was assessed using a thiobarbituric acid reactive substances (TBARS) lipid peroxidation assay kit (DoGenBio, Seoul, Republic of Korea). Cell viability was expressed as a percentage relative to the untreated control cells.
NGF-induced differentiated cells exposed to LPS in 6-well plates (2 × 10⁵ cells/well) were treated with flavonoid compounds (0, 5, and 25 μM) for 48 h. Lipid peroxidation and acetylcholinesterase activity were measured using TBARS and acetylcholinesterase (AChE) assay kits (DoGenBio, Seoul, Republic of Korea), respectively. The levels of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1β were determined using enzyme-linked immunosorbent assay (ELISA kits, R & D Systems, Minneapolis, MN, USA, and Abcam, Cambridge, MA, USA, respectively).

Phenolic compounds were identified through comparisons with retention times of pure standards: gallic, chlorogenic, caffeic, p-coumaric, sinapic, t-cinaminic acids, quercetin-3-O-rutinoside, kaempferol-3-O-glucoside, luteolin, quercetin, apigenin, and kaempferol (Sigma-Aldrich, Warsaw, Poland), each possessing 99.9% purity. Standard solutions were prepared from these pure compounds, with five injections performed for each standard. Chromatographic peak areas were recorded and calculated based on standard solution concentrations (Figures S1 and S2). From the obtained standard curves (Figures S3 and S4), mathematical equations were derived. Using these equations and dilution coefficients, the concentrations of individual phenolic compounds were determined.

Cell lysates were obtained from 3T3-L1 or HepG2 cells. Cells were scraped and lysed with M-PER lysis buffer. After centrifugation for 15 min at 16,000×g, the supernatant was obtained, and protein content was quantified using Bradford reagent. Before drug treatment, the samples were diluted to achieve a protein concentration of 1 mg/mL. Samples were treated with the Kaem or DMSO for 2 h at 25 °C and then incubated with pronase (5, 10, and 20 µg/mL) or distilled water for 10 min at 25 °C. After the reaction, SDS was added to the sample and the samples were heated at 100 °C. A portion of each sample was used for LC–MS/MS analysis. Sample preparation and proteome analysis were conducted as indicated in the previous publication⁶⁹ . For western blot analysis, VDAC1 or Na⁺K⁺ ATPase was used as an internal control. For the structure–activity-relationship (SAR) analysis, kaempferol (Sigma-Aldrich, 60010), Acacetin (Sigma-Aldrich, 00017), isosakuranetin (Sigma-Aldrich, PHL82569), Biochanin A (Sigma-Aldrich, D2016), (−)Epicatechin (Sigma-Aldrich, E4018), Genistein (Sigma-Aldrich, G6649) were used.

In this study, most of the chemicals, reagents, and standards were analytical grade and purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Gallic acid, L-ascorbic acid, vanillin, hexahydrate aluminium chloride, Folin-Ciocalteu’s phenol reagent, sodium phosphate, iron(III) chloride hexahydrate (Fe[III]Cl₃.6H₂O), hydrated sodium acetate, hydrochloric acid, sodium carbonate anhydrous, ammonium molybdate, quercetin, catechin, 2,2′-diphenyl-1-picrylhy-drazyl (DPPH), 2,4,6tripyridyl-s-triazine (TPTZ), and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from the Sigma-Aldrich (Castle Hill, NSW, Australia) for the estimation of polyphenols and antioxidant potential. Sulfuric acid (H₂SO₄) with 98% purity was purchased from RCI Labscan (Rongmuang, Thailand). HPLC standards including Gallic acid, p-hydroxybenzoic acid, caftaric acid, caffeic acid, protocatechuic acid, sinapinic acid, chlorogenic acid, syringic acid, ferulic acid, coumaric acid, catechin, quercetin, quercetin-3-galactoside, diosmin, quercetin-3-glucuronide, epicatechin gallate, quercetin-3-glucoside, kaempferol and kaempferol-3-glucoside were produced by Sigma-Aldrich (Castle Hill, NSW, Australia) for quantification proposes. HPLC and LC-MS grade reagents including methanol, ethanol, acetonitrile, formic acid, and glacial acetic acid were purchased from Thermo Fisher Scientific Inc. (Scoresby, VIC, Australia). To perform various in vitro bioactivities and antioxidant assays, 96 well-plates were bought from the Thermo Fisher Scientific (VIC, Australia). Additionally, HPLC vials (1 mL) were procured from the Agilent technologies (VIC, Australia).

ReagentQuercetin, kaempferol, potassium oxonate, xanthine, nicotinamide adenine dinucleotide (NAD⁺), uric acid, allopurinol, tetraethoxypropane (TEP), trichloroacetic acid (TCA), 2-thiobarbituric acid (TBA) and bicinchoninic acid kit were purchased from Sigma-Aldrich Chemical Co. (Steinheim, Germany). All other reagents were purchased from Merck (Darmstadt, Germany). The reagents used were from of analytical grades. Parsley (Petroselinum crispum) leaves were collected from a same vegetable garden, Tabriz, Iran. The following study was conducted in the Department of Nutrition and Biochemistry, Tehran University of Medical Sciences, Iran, between May 2007 and October 2008.
Test compound preparationParsley (Petroselinum crispum) leaves were carefully washed with water and left to dry at room temperature. Then they were weighted and completely blended in distilled water (1 : 1 w/v). Quercetin and kaempferol were dissolved in propylene glycol (17 (link)). allopurinol used as a positive control, was prepared in 0.9% saline. All freshly prepared juicy samples were administrated to the corresponding groups by oral gavage once a day for 2 weeks.
AnimalsA total of 60 male Wistar rats (body weights: 180-200 g) were obtained from the animal house of Tabriz University of Medical Sciences, Iran. They were fed with a commercial laboratory diet and allowed food and water ad libitum for an acclimatization period of 1 week prior to the experiment. All animals were maintained on a 12 h/12 h light/dark cycle and the temperature and humidity were kept at 18 ± 1°C and 50%, respectively. They were handled according to the recommendation of the local and national ethic committees.
Animal model of hyperuricemia in ratsExperimentally-induced hyperuricemia in rats (due to inhibition of uricase with potassium oxonate) was used to study antihyperuricemic and antioxidant effects of test compounds (16 (link)). Briefly, 250 mg/Kg, uricase inhibitor, potassium oxonate (PO), dissolved in 0.9% saline solution was administrated intraperitoneally to each animal 1 h before oral administration of test compounds.
Exprimental designThe animals were randomly divided into ten equal groups (n = 6). group 1: untreated, non- hyperuricemic animals; group 2: normal animals given 5 g/Kg parsley; group 3: normal animals given 5 mg/Kg kaempferol; group 4: normal animals given 5 mg/Kg quercetin; group 5: normal animals given 5 mg/Kg allopurinol; group 6: hyperuricemic animals; group 7: hyperuricemic animals given 5 g/Kg parsley; group 8: hyperuricemic animals given 5 mg/Kg kaempferol; group 9: hyperuricemic animals given 5 mg/Kg quercetin; group 10: hyperuricemic animals given 5 mg/Kg allopurinol.
Sample preparationBlood sample was taken from each rat by cutting the tail tip 1 h after the test compound administration at first, 7^th and 14^th days of the study. Serum was obtained by centrifuging blood sample at 6000 rpm for 10 min. For HPLC analysis, the serum was filtered using SPARTAN 13/0.45 RC, Watman. The sera were stored at -20°C until use. At the end of the experiment, rats were anesthetized between 09.00 and 10.00 am. Their livers were removed, weighed and then rapidly washed in cold saline (0.9%) and placed in ice-cold isotonic potassium chloride solution (1.15% KCl w/v) containing 0.1 mM EDTA. The livers were then chopped into 4-5 volumes of 50 mM phosphate buffer (pH 7.4) and homogenized by a homogenizer fitted with a Teflon pestle. The homogenate was then centrifuged at 3000 g for 10 min, the lipid layer was carefully removed, and the resulting supernatant fraction was further centrifuged at 15,000 g for 60 min at 4°C. The supernatant was stored at -20°C until use. At the end of the experiment, rats were anesthetized between 09.00 and 10.00 am. Their livers were removed, weighed and then rapidly washed in cold saline (0.9%) and placed in ice-cold isotonic potassium chloride solution (1.15% KCl w/v) containing 0.1 mM EDTA. The livers were then chopped into 4-5 volumes of 50 mM phosphate buffer (pH 7.4) and homogenized by a homogenizer fitted with a Teflon pestle. The homogenate was then centrifuged at 3000 g for 10 min, the lipid layer was carefully removed, and the resulting supernatant fraction was further centrifuged at 15,000 g for 60 min at 4°C. The supernatant was stored at -80°C till the use time (18 (link)-20 (link)).
Uric acid determinationThe serum uric acid levels were analyzed by the high performance liquid chromatography (HPLC) method using a system supplied by Waters Associates, Northwich, Cheshire which consisted of a Waters 515 pump, Waters 717 plus Autosampler, Waters 2487, Dual λ Absorbance Detector. The mobile phase was a mixture of 100 mM KH₂PO₄ (pH 3.5): Methanol (97:3, v/v).
Separations were performed on a C-18 column (Perfectsil Target ODS-3 (5 μM), 250´ 4.6 mm) with a C-18 guard column (Perfectsil Target ODS-3 (5 μM), 10´ 4 mm). The effluent was monitored by UV detection at 290 nm at a flow rate of 1.0 mL/min. Standard solutions of uric acid in the range of 0.1 to 20 mg/dL were prepared in mobile phase. Serum uric acid concentrations were expressed as mg/dL. 6-Mercaptopurine (1 mM) was used as the internal standard (21 (link)).
XO and XDH activity determinationThe XO and XDH activity were measured spectrophotometrically by monitoring the production of uric acid from xanthine according to Prajda and Weber’s method (22 (link)). In the case of XDH, the assay mixture consisted of 50 mM phosphate buffer (pH 7.4), 200 µM NAD⁺, and 100 µL of the enzyme solution. After preincubation at 37°C for 15 min, the reaction was initiated by the addition of the substrate solution. After 30 min, the reaction was terminated by adding 0.5 mL HCl (0.6 M), and the absorbance was measured at 290 nm using a Shimadzu 2550 UV/VIS spectrophotometer which was controlled by the Shimadzu UV Probe personal software package including kinetics software. The instrument was connected to a Shimadzu cell temperature control unit. XO activity was measured using a similar method described for XDH with the difference being that molecular oxygen was used in place of NAD⁺ as electron acceptor. One unit (U) of activity was defined as 1 nmole of uric acid formed per min at 37°C, pH 7.4.
Protein determinationProtein concentration was determined spectrophotometrically by bicinchoninic acid kit using bovine serum albumin as the standard.
Total antioxidant capacity assayThe total antioxidant capacity of serum was determined by measuring its ability to reduce ferric ions (Fe³⁺) to ferrous form (Fe²⁺) by the FRAP (Ferric Reducing Ability of Plasma) test. The FRAP assay measures the change in absorbance at 593 nm owing to the formation of a blue colored Fe²⁺-tripyridyltriazine compound from Fe³⁺ by the action of electron donating antioxidants. The FRAP reagent consists of 300 μmole/mL acetate buffer (pH 3.6), 10 μmolee/mL tripyridyltriazine (TPTZ) in 40 μmole/mL HCl and 20 μmole/mL FeCl₃ in the ratio of 10:1:1. Briefly, 30 μL of serum was added to 1.0 mL freshly prepared and prewarmed (37°C) FRAP reagent in a test tube and incubated at 37°C for 10 min. The absorbance of the blue colored complex was read against a reagent blank (1.0 mL FRAP reagent + 30 μL distilled water) at 593 nm. Standard solutions of Fe²⁺ in the range of 10 to 1000 μmole/L were prepared from ferrous sulphate in water. FRAP values were expressed as μmole Fe³⁺ reduced to Fe²⁺ per liter (23 (link)).
Lipid peroxide determinationLipid peroxide in the serum was measured using Yoshioka method (24 (link)). Briefly, 0.5 mL serum was shaken with 2.5 mL of 20% trichloroacetic acid (TCA) in a 10 mL centrifuge tube. 1 mL of 0.67% TBA was added to the mixture, shaken, and warmed for 60 min in a boiling water bath followed by rapid cooling. Then it was shaken into a 4 mL of n-butanol layer in a separation tube and malondialdehyde (MDA) content in the serum was determined at 532 nm by spectrophotometer against n-butanol. The standards of 0.1 to 20 μmole/L tetraethoxypropane (TEP) were used. The results were expressed as μmole/L serum.
Statistical analysisAll the samples and standards were run in duplicate and the results were expressed as mean ± SD. The statistical comparison between the experimental groups was performed by independent-sample t-test using SPSS computer program. The probabilities of 5% or less (p ≤ 0.05) were considered significant.