Plant Samples
Plants were collected from a field site at the Teaching Base of USAMVB Timisoara. The two types of plant specimens, the variety Petran (P1) and Plamen (P2) were collected and tagged on the spot using standard techniques (the varieties were grown under the same conditions).
The plant specimens that were preserved as herbarium specimens were identified using the standard reference for this purpose [42], compared with specimens from the Department of Plant Resources, and identified with the help of taxonomists from the Department of Plant Phytotechny at the USAMVB Timisoara.
Extraction of Active Biologic Compounds
The four solvents used for extraction were methanol (80%), ethanol (96%), isopropanol (100%) and ethanol (60%). The general process of maceration on a small scale involved placing suitably crushed plant material, or a moderately coarse powder made from it, in a closed vessel and adding the selected solvent to allow the solvent sufficient time to penetrate the cell wall to solubilize the constituents within the cells and for the resulting solution to penetrate the cell wall outwards. As the system is static, except for the occasional shaking, the process of extraction works by molecular diffusion, which is very slow. A closed vessel is used to prevent evaporation of the solvent during the extraction period, and thus avoids batch-to-batch variation.
At the end of the maceration process, when equilibrium has been reached, the solution is filtered by passage through a special press.
The concentrations of active constituents in the strained and expressed liquids, sometimes called miscella, are identical, and can thus be combined. Given that the expressed liquid may be cloudy with colloidal and small particles, a certain amount of time is often necessary for coagulation and settling of particulate matter.
The settled matter is filtered using a filter press or any other suitable apparatus.
The maceration extraction method is the simplest non-selective method. C. officinalis flowers were kept in a cold environment in contact with the solvent at room temperature (17-22°C) for 14 h, stirring occasionally before the extract was separated. An additional round(s) of maceration increased the efficiency of the process. The extracted product was first mixed with 1/2-2/3 of the total quantity of solvent, after which the liquid was separated and the residue released.
It comes in contact with the rest of the solvent, resulting in one ninth quantity of solution mining. The two liquids extract and filter together after another 24 hours.
Owing to the relatively reduced thermal stability of natural aromatic compounds, the process was conducted in the presence of the antioxidant ascorbic acid (0.5% w/v) [43]. Extraction rate (1: 10 g of flowers powder/mL solvent).
High Performance Liquid Chromatography (HPLC)
Polyphenol-containing extracts of C. officinalis flowers were separated on a C18 (150 mm × 4.6 mm, 3 μm) column, with a mobile phase consisting of three different solvents, using an Agilent 1100 HPLC apparatus. The flow rate was 0.7 mL/min and the injection volume was 20 μL. For the first separation system (ternary solvent), which was used for the separation of flavanols and phenolic acids, the initial mobile phase consisted of 2% acetic acid in water/methanol. For separation of flavonols the second system (binary solvent: 0.25 mM phosphate buffer, pH = 2.5/acetonitrile), polyphenolic compounds were separated using a third system (binary solvent: 0.1% formic acid in water/methanol). The column temperature was adjusted at 20°C.
Phenolic compounds were detected at 260 nm. The separated compounds were identified by comparing their retention time (Rt) and UV spectra with those of authentic standards. Compounds were quantified using a standard calibration curve.
Total phenolic and flavonoid contents were quantified as the sum of the related identified phenolic compounds. The compounds were identified by comparing with standards of each identified compound using Rt, the absorbance spectrum profile, and also by running the samples after the addition of pure standards [35].
Total polyphenol content
The total polyphenol content was calculated using the FC reagent and Na2CO3 10%; ulterior the maximum absorption was evaluated by the obtained coloration, in relation to the calibration curve. The total content of phenolic compounds was evaluated using colorimetry based on the chemical reduction of the mixture of tungsten and molybdenum oxides to form a blue product that absorbs strongly at 760 nm.
The formation of blue compounds derived from FC-reactive phenols was independent of the structures of the phenolic compounds, enabling quantification of the full complement of phenolics from plant extracts. Approximately 10 g of pulverized plant material was extracted with 100 mL solvent. The total concentration of phenolic compounds in the extract was calculated through comparison with a curve prepared in a similar way based on measurement made using known concentrations of GA ranging between 0-00 μg GA/100 mL. The total content of phenols in samples was expressed in equivalents of GA (mg) in a gram of dried plant material.
Levels of polyphenols were expressed in mg/g equivalents of GA in relation to a final concentration 20 μg/mL.
Total flavonoid content
The combined level of all flavonoids was calculated by reaction with a 3% (w/v) solution of AlCl3 (anhydrous salt) prepared in ethanol.
Concentrations were correlated with absorption values by using a calibration curve constructed using known concentrations of QE. The total content in flavonoids expressed in mg/g (QE equivalents) was related to a final concentration by 20 μg/mL.
Estimation of antioxidant activity using DPPH
Approximately 10 g of pulverized plant material was extracted in 100 mL of an 80% aqueous solution of ethanol (or other solvents) at the room temperature for 1 h.
The extracts were filtered, and the filters were left to evaporate in a dry environment.
The percentage free radical scavenging activity was determined using the formula (1), where Ai was the absorption before the tested extract adding and Af was the absorption value after a 5 min reaction time:
The neutralization effect of the DPPH◦ free radical was calculated at three different concentrations of methanol and ethanol extracts: portions of 0.05 mL from extracts of 10, 5 and 2.5 mg/mL were mixed with 2.95 mL solution DPPH◦ in the cuvette used for spectrophotometry.
After a 5-min reaction time, the absorption at 420 nm was compared with that of methanol. QE was used as a positive control. At a concentration of 2.5 mM, QE was able to fully neutralize (the level of DPPH◦ radical used).
Ferric Reducing Antioxidant Power (FRAP) assays
We used a simple spectrophotometric method to evaluate the antioxidant power of the plant extracts. This involved reduction of ferric tripyridyltriazine [Fe(III)-TPTZ] to coloured ferrous tripyridyltriazine [Fe(II)-TPTZ] at low pH.
The coloured product absorbs strongly at 593 nm.
Pigment Analysis
All samples were extracted in acetone. The pigments were extracted from C. officinalis flower with 80% acetone. We used the specific absorption coefficients of chlorophylls a and b, and carotenoids (Car) to estimate their levels using spectrophotometry. All of the chlorophylls contained two main absorption bands of radiations: one on the blue side or near the UV portion of the electromagnetic spectrum, and the other on the red side or near the IR region of the electromagnetic spectrum. The lack of a significant absorption on the green side of the spectrum confers on chlorophylls their characteristic green or blue-greenish colors.
This method determines the amount (mg) in 100 mL of carotenoid extract. The pigment compounds calculated according to Equations 2, 3 and 4:
(2)
(3)
(4)
The working methods were based on the Ro Pharmacopeia 1993 techniques in conformity with international standards (Ph. Eur. Reference Standards and WHO, ICRS, 2010, [44–47].
Reagents and Other Materials
QE and GA were obtained from Sigma-Aldrich (St. Louis, MO, USA), whereas all other reagents were obtained from Merck. All chemicals and reagents were of analytical grade.
Statistical Analysis
All the results of spectrophotometric determinations of flavones and polyphenols were reproduced after establishing the confidence interval calculated for an average of a minimum of five determinations. The statistical differences between the means were determined using a two-tailed paired Student's t-test.
