Corresponding author: Petya Koleva (
Pharmacia 68(4): 819–826
DOI
Bulgarian medical herbs are known worldwide for their high content of biologically active substances. They are rich in various chemical compounds – alkaloids, glycosides, saponins, polysaccharides, polyphenols, coumarins, essential and fatty oils, vitamins, flavonoids and more. App. 500–600 medicinal plants are used in Bulgarian folk medicine. App. 300 species are most often used for the needs of the pharmaceutical industry, phytotherapy and for export. Around 12,000 species of medicinal plants are used worldwide, of the currently known about 500,000 species of higher plants or only 2.4% of the world’s flora are known and used as medicinal plants.
Polyphenols, including the flavonoids group, are secondary metabolites in higher plants, and more than 8,000 compounds are currently known. They are present in various parts of plants that are used for food, as well as in a number of medicinal plants. It is interesting to note that half of the polyphenolic compounds belong to the group of flavonoids and are found as aglycones, glycosylated forms and methylated derivatives (Kumar and Padney 2013;
It is known that fruits and vegetables are a very rich source of flavonoids from the group of catechins and the flavonol quercetin (Tsanova-Savova et al. 2003,
The aim of the study is to determine the content of polyphenols in selected Bulgarian medicinal plants and food supplements, present at Bulgarian market, by developing a validated HPLC method for determination the individual representatives of catechins – (+)-catechin and (-)-epicatechin and of the flavonol – rutin. Furthermore for overall characterization of their antioxidant polyphenolic content, the selected samples are analyzed for Total phenolic and Total flavonoids with validated spectrophotometric methods.
Medical plant dry material (100 g) was purchased from herbal pharmacy, and food supplements were purchased from pharmacy net in Sofia, according their wide application at Bulgarian market. The selected samples are described in Table
Description of samples.
Samples | |
---|---|
|
|
Elderberry, fruit |
|
Cranberry, fruit |
|
Hawthorn, fruit |
|
Wild mint, leaves |
|
Lemon balm, leaves |
|
|
|
Elderberry (1) | Elderberry, Syrup, containing extract of |
Elderberry (2) | Elderberry, Syrup, containing standardized extract of |
Cranberry | Cranberry, Capsules, containing dry extract of |
Lemon balm | Lemon balm, Tablets, containing 300 mg |
Mint, Hawthorn, |
Mint, Hawthorn, |
Hawthorn | Hawthorn, Tablets, containing 325 mg |
The powdered material of plant or food supplements (0.2500 g) were weighted into a 25 ml volumetric flask of and extracted with 80% methanol/water (w/w) in an ultrasonic bath for 30 min at 30 °C. The solutions were ultracentrifuged at 10000 rpm for 5 min and filtered through a membrane filter (0.45 µm) prior to analysis. The extraction solution was selected according our previous studies and literature data (Tsanova-Savova et al. 2003;
(+)-Catechin and Gallic acid were purchased from Sigma (St. Louis, MO). (-)-Epicatechin and Rutin trihydrate, Folin – Ciocalteau reagent, was obtained from Alfa Aesar (Thermo Fisher Scientifics, Kandel, Germany). Acetonitrile, methanol and water were HPLC grade and were obtained from Macron Fine Chemicals (Avantor, Glivice, Poland), and all other chemicals and reagents were purchased from Alfa Aesar (Thermo Fisher Scientifics, Kandel, Germany).
Folin – Ciocalteau reagent was used to determine the total phenols, which oxidizes the phenolates to a blue complex, which is determined at λ = 750 nm. The absolute calibration method was used to calculate the amount of Total phenolic. The determination was performed according
Total flavonoids were determined, according
Spectrophotometric measurement of the absorption of Total phenolic compounds and Total flavonoids was performed on a Spectrophotometer, Lanbda 25, Perkin Elmer.
The HPLC system consisted of a Perkin-Elmer (Norwalk, CT) Flexar LC pump, Flexar Photo Diode Array Plus detector (
Chromatographic separation of phenolic compounds - (+)-Catechin, (-)-Epicatechin and Rutin, was carried out using Luna C18 column (3 μm, 150 mm × 4.6 mm, Phenomenex, USA), equipped with precolumn. Elution was performed at a flow rate of 0.9 ml/min. The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). A linear gradient program was applied as follows: 0–2 min, 15% B; 2–4 min, 20% B; 4–6 min, 25% B; 6–8 min, 30% B; 8–10 min, 35% B; 10–12 min, 35% B; 5 min, re-equilibration of the column with 15% B. The column temperature was constant 30 °C. The injection volume of the sample solution was 20 µl. The detection wavelength for (+)-catechin and (-)-epicatechin was 280 nm, for rutin was 355 nm, and the reference wavelength was set at 620 nm. Analog output channel A at 355 nm and analog output channel B at 280 nm both with bandwidth 19 nm were fixed.
The identification of chromatographic peaks was achieved by comparing the retention time of the eluted peaks and comparing peak shape at two channels with different wavelength (280 and 355 nm). For quantitative analysis, a calibration curve was obtained by injection of known concentration of standard solutions.
Stock solutions (1 mg/ml) of analytes (+)-catechin, (-)-epicatechin, rutin, gallic acid were prepared from pure compounds by dissolving each compound in methanol. Working standard solutions were prepared daily from stock solutions by dilution with appropriate volume of 80% methanol. All solution were stored at 4 °C and used within five days.
The values of limit of detection (
The linearity of the response was evaluated by analysis of standard solutions of (+)-catechin, (-)-epicatechin and rutin for HPLC analysis; of gallic acid for Total phenolic determination and rutin for Total flavonoids determination. Triplicate measurement was made for each standard solution. Calibration curves, correlation coefficients and linearity range are presented in Table
Linearity,
Compound | Calibration curve | Correlation coefficient R2 (n = 3) | Linear range (μg/ml) | RT, min | |||
---|---|---|---|---|---|---|---|
(+)-Catechin | y = 11187x - 1240.8 | 0.9996 | 0.20–30.00 | 4.41 | 0.20 | 0.12 | 0.40 |
(-)-Epicatechin | y = 12925x - 2637 | 0.9993 | 0.20–30.00 | 6.09 | 0.26 | 0.12 | 0.40 |
Rutin | y = 12925x - 2637 | 0.9997 | 0.02–40.00 | 9.67 | 0.09 | 0.02 | 0.07 |
Linearity,
Calibration curve | Correlation coefficient R2 (n = 3) | Linear range (μg/ml) | |||
---|---|---|---|---|---|
Total phenolic | y = 0.1x + 0.0088 | 0.9991 | 0.21–7.22 | 0.10 | 0.21 |
Total flavonoids | y = 0.0131x + 0.0014 | 0.9959 | 0.47–18.75 | 0.25 | 0.47 |
The precision of both HPLC method and spectrophotometric methods for determination of Total phenolic and Total flavonoids was assessed at two levels by multiple analysis of a standard solution (2 μg/ml). At first level the intra-day precision of repeatability was determined within the same day in a single analysis on a single instrument. The second level was the inter-day precision or reproducibility and was determined over three days on the same instrument. The obtained results were expressed as relative standard deviation (
The recovery was evaluated by adding measured amount of pure standards to a sample of
Repeatability. Reproducibility and Accuracy of determination of (+)-catechin. (-)-epicatechin and rutin and of Total phenolic and Total flavonoids.
Accuracy | ||||||
---|---|---|---|---|---|---|
Inter-Day (n = 6) | Intra-Day (n = 6) | Inter-Day (n = 6) | Intra-Day (n = 6) | (Analytical Recovery. %) (n = 3) | ||
|
||||||
(+)-Catechin | 2.59 | 2.72 | 3.56 | 4.75 | 99.53 | |
Cranberry. Supplement | 97.41 | |||||
(-)-Epicatechin | 2.00 | 2.35 | 1.94 | 3.53 | 92.46 | |
Cranberry. Supplement | 93.57 | |||||
Rutin | 2.17 | 1.39 | 1.24 | 2.28 |
|
96.02 |
Cranberry. Supplement | 95.97 | |||||
|
1.98 | 3.06 | 2.04 | 3.78 | 93.15 | |
Cranberry. Supplement | 95.34 | |||||
|
1.51 | 2.72 | 2.40 | 4.12 | 96.57 | |
Cranberry. Supplement | 97.13 |
1 Repeatability and Reproducibility of Standard Solution (2 μg/ml). 2 Repeatability and Reproducibility of
The results of HPLC analysis enabled the identification of (+)-catechin, (-)-epicatechin, and rutin within 12 min. The HPLC chromatograms of standard solutions and
The chromatograms show a very good baseline resolution of analytes. The use of formic acid in the mobile phase suppresses the strong specific interactions between the sorbent (residual silanol groups) and the sorbate (phenolic groups in the analytes) due to orientational interactions between molecules with permanent dipoles. Furthermore, to eliminate such interactions, for HPLC separation of flavonoids, the use of octadecylsilane reverse phase columns, with no further endcapping of the residual silanol groups, results in a large tail of the picks. In the present study, the use of a Luna column, in combination with the reduced size of the sorbent (3 µm) and the suppression of the ionization of the analyte by proper selection of the mobile phase, provide symmetrical peaks in the chromatogram.
Chromatogram of Standard mix of (+)-catechin, (-)-epicatechin and rutin at 275 nm (
Chromatogram of
Chromatogram of
The limits of quantification were 0.4 µg/ml for (+)-catechin and (-)-epicatechin, and 0.07 µg/ml for rutin (Table
Polyphenols content in selected medical plants and food supplements.
|
Total phenolic | Total flavonoids | (+)-Catechin | (-)-Epicatechin | Rutin |
---|---|---|---|---|---|
mg |
mg |
µg/g | µg/g | µg/g | |
16.88 | 16.24 | 149.6 | – | 2818.7 | |
18.42 | 17.41 | 260.1 | 504.51 | 25.9 | |
12.49 | 7.08 | – | 363.2 | 36.8 | |
67.38 | 54.59 | – | – | 7332.5 | |
65.17 | 57.76 | – | – | 402.5 | |
|
|||||
Elderberry (1) | 5.16 | 3.60 | – | – | 51.5 |
Elderberry (2) | 20.76 | 18.76 | 102.1 | – | 262.3 |
Cranberry | 20.92 | 20.25 | 161.4 | 101.1 | 41.3 |
Lemon balm | 32.30 | 15.16 | – | – | 145.7 |
Mint. Hawthorn. |
21.54 | 8.10 | 266.5 | 357.6 | 1214.7 |
Hawthorn | 28.76 | 9.95 | – | 618.1 | 26.2 |
(-)- lower than |
The results for limit of determination for Total phenolic and Total flavonoids measurements are 0.21 µg
The data for repeatability. reproducibility and accuracy of methods applied is presented in Table
These data support the suitability of the methods for its application to real samples.
The results of analysis of the selected medical plants and food supplements in this study show that among medical plants studied
The results of the selected food supplements show that their polyphonic load is in general lower than those of individual medical plants analyzed. It should be noted the great variability of results of Elderberry Syrup, containing unstandardized and standardized extract of
Our results for Total phenolic and Total flavonoids in Lemon balm (
It is known that medicinal plants and fruits are widely used in folk medicine are a rich source of antioxidant polyphenols and in particular flavonoids. At the same time, a rich palette of food supplements is currently available, with claims to powerful antioxidant activity. Furthermore, quantitative data for their evaluation are sporadic and unsystematic. In this regard, we have of characterized the quantitative content of Total phenolic compounds, Total flavonoids and the individual representatives of catechins – (+)-catechin and (+)-epicatechin, as well as the flavonol rutin with a validated HPLC method in a 5 of Bulgarian medicinal plants and fruits, which are widely used for the preparation of herbal infusions and decoctions and in 5 food supplements. The results show that
The present study is with financial support of Medical Science Council of Medical University-Sofia. Contract Nr. D-127/24.06.2020 – Project “Comparative study of antioxidant polyphenolic content of medical plants. their fruits and food supplements”. Grant 2020.