Chemicals and reagents
Dulbecco’s Modified Eagle Medium (DMEM), DQ gelatin and penicillin-streptomycin were supplied from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was supplied from Thermo Scientific (USA). Sirius Red/Fast Green Collagen Staining Kit was purchased from Chondrex, Inc. (Redmond, WA, USA). Sulforhodamine B reagent, hyaluronicacid and mushroom tyrosinase were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Plant materials
C. fistula flowers were obtained from Lampang Herb Conservation, Lampang Province, Thailand. A voucher specimen number (023197) was certified by Wannaree Charoensup, Botanist at the herbarium of the Flora of Thailand, Faculty of Pharmacy, Chiang Mai University, Thailand.
Preparation of Cassia fistula flower extract
C. fistula flowers were dried in an airy room and then 500 g of dried flowers were finely ground into powder. After that, the powder was soaked in 50% ethanol and then shaken at 70 rpm for 24 h. After 24 h, the samples were then filtered through filter paper to separate the residue. The residue was soaked in 50% ethanol and shaken at 70 rpm for 24 h and this step was repeated 2 times. The filtrated samples were pooled together and evaporated by a rotary vacuum evaporator (BUCHI, Switzerland) to obtain the water fractions. Hexane was added to the water fraction at a ratio of hexane 2:1. The samples were shaken and allowed to separate over 30 min. The samples were separated into two fractions, which were hexane and water fractions. Then, the water fraction was collected and 10% charcoal was added for a 30 min period with mild stirring at room temperature. The samples were filtered through filter paper and celite to separate the charcoal. The samples were mixed with saturated butanol at a ratio of saturated butanol 2:1 and the samples were allowed to separate over a 12 h period and this step was repeated 3 times. The samples were separated into two fractions, which included water and butanol fractions. Water was further added to the butanol fractions to an equal volume and these fractions were allowed to evaporate through the use of a rotary vacuum evaporator. The evaporated samples were freeze-dried to obtain the C. fistula flower extract powder.
Quantification of total phenolic content in C. fistula flower extract using UV-visible spectrophotometer
Total phenolic content in C. fistula flower extract was determined using the Folin-Ciocalteu assay. Briefly, 0.4 mL of C. fistula flower extract were mixed with 0.3 mL of 10% Folin-Ciocalteau reagent and incubated in a dark at room temperature for 3 min. Then, 0.3 mL of sodium carbonate solution was added and the solution was further incubated in the dark at room temperature for 30 min. The absorbance was evaluated at 765 nm using a UV-visible spectrophotometer. The concentration of the total phenolic content was calculated and compared with a standard curve for gallic acid (GA) at 0–20 μg/mL. The total phenolic content was reported as milligrams of GA equivalents per gram of C. fistula. flower extract (mg GAE/g extract).
Quantification of phenolic compounds in C. fistula flower extract using HPLC analysis
Various types of phenolic compounds in C. fistula flower extracts were analyzed using HPLC analysis and were then compared with standard gallic acid, catechins, protocatecheuic acid, vanillic acid, chlorogenic acid, ferulic acid and coumaric acid. The flower extracts were dissolved in 50% ethanol and were further assessed by HPLC (Agilent Tecnologies, CA, USA) using reversed-phase C18 column (WATER, MA, USA). The mobile phase consisted of methanol (A) and 0.1% trifluoroacetic acid (TFA) in water (B) with gradient condition. The flow rate was set at 1.0 mL/min and the detection wavelength was recorded at 280 with a UV detector. The concentration levels of the phenolic compounds were calculated and compared with the standard curve considering the standard concentrations and peak areas (mg/g extract).
Quantification of total flavonoid content in C. fistula flower extract using colorimetric assay
Total flavonoid content was determined by modified aluminium chloride (AlCl3) colorimetric assay as was previously described [22]. Briefly, 0.25 mL of flower extract were mixed with 0.125 mL of 5% sodium nitrite (NaNO2) and the solution was incubated for 5 min at room temperature. Then, 0.125 mL of 10% AlCl3 were mixed into the mixture and it was kept in the dark for 5 min. After which, 1 mL of sodium hydroxide (NaOH) was added and the solution was incubated for 15 min at room temperature in the dark. The absorbance was measured at 510 nm using a UV-visible spectrophotometer. The total flavonoid content was calculated and compared with the standard catechin values and expressed as mg catechin equivalent per gram of extract (mg CE/g extract).
Cell cultures
Primary human skin fibroblasts were aseptically isolated from an abdominal scar after a surgical procedure involving a cesarean delivery at the surgical ward of CM Maharaj Hospital, Chiang Mai University (Chiang Mai, Thailand) (Study code: BIO-2558-03549 approved by Medical Research Ethics Committee, Chiang Mai University). Fat was removed from the starting material and it was soaked in DMEM containing anti-penicillin and streptomycin. After that, the skin was immersed in DMEM containing 1% protease (Dispase, Gibco, Grand Island, NY, USA) for 48 h at 4 °C. Epidermal layers were removed and the normal fibroblasts were isolated from the dermis. The fibroblasts were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin. The cells were maintained in a 5% CO2 humidified incubator at 37 °C.
Cell viability assay
The cell viability assay of C. fistula flower extract against skin fibroblast cells was measured using a sulforhodamine B (SRB) assay as was previously described [23]. Briefly, the cells (8x103cells/well) were seeded in a 96-well plate and incubated at 37 °C, 5% CO2 for 24 h. After that, the cells were treated with or without various concentrations (0–200 μg/mL) of C. fistula flower extract for 48 h. After 48 h, 10% (w/v) Trichloroacetic acid (TCA) was added to the cells and the cells were then incubated at 4 °C for 1 h. The medium was removed and the cells were rinsed with slow running tap water. 0.057% (w/v) SRB solution (100 μL) was added to each well and the cells were incubated for 30 min at room temperature. The SRB solution was removed and then the cells were washed 4 times with 1% (v/v) acetic acid and they were allowed to dry at room temperature. The dye was dissolved with 10 mM Tris base solution (pH 10.5) and the absorbance was measured at 510 nm using a microplate reader.
Collagen synthesis assay
Total intracellular collagen in the fibroblast cells was determined using Sirius Red/Fast Green Collagen Staining Kit (Chondrex, Inc, Redmond, WA, USA) according to the manufacturer’s instructions. Briefly, skin fibroblast cells were seeded in 24 well plates for 24 h at 37 °C, 5% CO2. After that, the cells were pre-treated with the serum free DMEM medium for 24 h. The medium was removed and the cells were then incubated with or without C. fistula flower extract (0-150 μg/mL) for 48 h. After 48 h, the cultured medium was removed. Then, the cells were washed with PBS and fixed with Kahle fixative for 10 min. The fixative was removed and the cells were washed with PBS. The Sirius Red/Fast Green dye solution was added to each well and the specimens were incubated for 30 min at room temperature. The dye was removed and the cells were washed four times with DI water or until the fluid was colorless. Finally, the extraction buffer was added and the absorbance was measured at 540 nm and 605 nm using a UV-visible spectrophotometer.
Collagenase activity assay
The collagenase activity was measured using modified fluorogenic DQ™-gelatin assay as has been previously described [24]. Briefly, various concentrations of C. fistula flower extract (0–200 μg/mL) were added in 96 well plates. One U/ml of collagenase was added in each well (100 μL/well). After that, 15 μg/mL of gelatin (DQ gelatin) was added and the mixtures were incubated for 10 min. The rate of proteolysis was determined by measuring the absorbance at 2 min intervals for 20 min using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. Enzyme activity was estimated by examining the slope of linear regression between time and absorbance over a 2-6 min period.
MMP-2 activity assay
The MMP-2 activity was determined by gelatin zymography as was previously described [25]. Fibroblast cells were cultured in DMEM serum-free medium for 24 h. After that, the culture supernatant was collected. The culture supernatant was subjected to 10% polyacrylamide gels containing 0.1% w/v of gelatin under non-reducing conditions. The gels were washed twice with 2.5% v/v of Triton X-100 for 30 min at room temperature to remove SDS. The gel slab was cut into slices which corresponded to the lanes and then the slices were put into different tanks and were incubated with an activating buffer (50 mM Tris-HCl, 200 mM NaCl, 10 mM CaCl2, pH 7.4) containing various concentrations of C. fistula flower extract (0–200 μg/mL) at 37 °C for 18 h. After that, a strip of the gels was washed and stained with Coomassie Brillant Blue R (0.1% w/v) and then was destained in 30% methanol and 10% acetic acid. MMP-2 activity appeared as a clear band against a blue background. Digestion bands were quantitated by the Image J program.
Hyaluronic Acid (HA) synthesis assay by ELISA
The effect of C. fistula flower extract on HA synthesis was determined using an ELISA kit as previously described [26]. The fibroblast cells (5.0 × 104 cells/well) were seeded in 24-well-plates for 24 h at 37 °C, 5%CO2. The cells were pre-treated in serum free medium for 6 h and were then treated with or without various concentrations of C. fistula flower extract (0–200 μg/mL) for 48 h. The cultured medium was collected and HA synthesis was measured by ELISA. The absorbance was measured at 450 and 570 nm using a microplate reader. The HA concentration in the cultured supernatant acquired from the treated fibroblast cells was calculated and compared with the standard curve of HA.
Tyrosinase inhibition assay
Tyrosinase inhibition assay was determined as was previously described [27]. Various concentrations of C. fistula flower extract were added to a 96-well-plate. The reaction was carried out in a sodium buffer (pH 6.4) containing 100 U/mL mushroom tyrosinase. L-DOPA substrate (1 mM) was added into the reaction mixture and it was then incubated for 10 min at room temperature. The change of the absorbance at 490 nm was measured every 1.5 min for 15 min using a microplate reader. The percent inhibition of tyrosinase was calculated by the following formula:
$$ \mathrm{Tyrosinase}\ \mathrm{inhibition}\ \left(\%\right) = \left[\left(\mathrm{A}-\mathrm{B}\right)/\mathrm{A}\right] \times 100, $$
A = slope of control at 490 nm
B = slope of test at 490 nm
Antioxidant activity assay
The antioxidant activity of the C. fistula flower extract was determined using 1, 1-diphenyl-2-picrylhydrazyl (DPPH)-free radical activity, as previously described [28]. Various concentrations of the flower extract (0–100 μg/mL) were prepared in methanol. 1 mL of 0.002% of DPPH was added to 1 mL of the flower extract solution and the mixture was kept in the dark for 30 min. Absorbance was measured at 517 nm using a spectrophotometer. The % inhibition was calculated and compared with standard vitamin E (1–100 μg/mL) using the following formula:
$$ \mathrm{Percent}\ \mathrm{inhibition}\ \mathrm{of}\ \mathrm{DPPH}\ \mathrm{activity} = \frac{{\mathrm{A}}_{\mathrm{control}\ \mathrm{at}\ 517\ \mathrm{nm}}-{\mathrm{A}}_{\mathrm{sample}\ \mathrm{at}\ 517\ \mathrm{nm}}}{{\mathrm{A}}_{\mathrm{control}\ \mathrm{at}\ 517\ \mathrm{nm}}}\times 100 $$
The antioxidant activity of the C. fistula flower extracts was confirmed using ABTS assay, as was previously described [29]. ABTS [2, 2′- azino-bis (ethylbenzthiazoline-6-sulfonic acid)] radical cation was prepared by mixing 7 mM ABTS stock solution with 2.45 mM potassium persulfate (K2S2O8) (1/1, v/v). The mixture was incubated in the dark for 12–16 h until the reaction was completed. The assay was conducted on 990 μL of ABTS solution and 10 μL of the flower extract (0–4 μg/mL). After 6 min, the absorbance was recorded immediately at 734 nm using a spectrophotometer. The percent inhibition of ABTS activity of the flower extract was calculated using the following equation:
$$ \mathrm{Percent}\ \mathrm{inhibition}\ \mathrm{of}\ \mathrm{ABTS}\ \mathrm{activity} = \kern0.5em \frac{{\mathrm{A}}_{\mathrm{control}\ \mathrm{at}\ 734\ \mathrm{nm}}-{\mathrm{A}}_{\mathrm{sample}\ \mathrm{at}\ 734\ \mathrm{nm}}}{{\mathrm{A}}_{\mathrm{control}\ \mathrm{at}\ 734\ \mathrm{nm}}}\times 100 $$
Statistical analysis
All data are presented as mean ± standard deviation (S.D.) values. Statistical analysis was analyzed by Prism version 6.0 software using one-way ANOVA with Dunnett’s test. Statistical significance was determined at * p < 0.05, ** p < 0.01, ***p < 0.001 or **** p < 0.0001.
