Corresponding author: Petya Koleva ( p.koleva@mc.mu-sofia.bg ) Academic editor: Plamen Peikov
© 2021 Petya Koleva, Silvia Tsanova-Savova, Slaveyka Paneva, Stefan Velikov, Zaharina Savova.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Koleva P, Tsanova-Savova S, Paneva S, Velikov S, Savova Z (2021) Polyphenols content of selected medical plants and food supplements present at Bulgarian market. Pharmacia 68(4): 819-826. https://doi.org/10.3897/pharmacia.68.e71460
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Background: Medicinal plants are a rich source of antioxidant polyphenols and in particular flavonoids.
Materials and methods: In the present study 5 Bulgarian medical plants and 5 food supplements, present at the Bulgarian market, are analyzed for their Total phenolic, Total flavonoids and selected individual flavonoids content. A HPLC method was developed and validated for simultaneous determination of (+)-catechin, (-)-epicatechin and rutin in plant and supplements, using PDA detection.
Results: The results show that polyphenols in the selected food supplements are lower than those of the medical plants analyzed. Mentha piperita and Melissa officinalis have the highest polyphenols content (67.38 and 65.17 mg GAE/g; 54.59 and 57.76 mg RE/g respectively). Rutin was detected in all samples analyzed, reaching highest levels in Mentha piperita (7332.5 µg/g), followed by fruits of Sambucus nigra (2818.7 µg/g).
Conclusion: The results of the study are a practical contribution to a more complete characterization of the polyphenolic composition in Bulgarian medicinal plants.
Catechins, HPLC, Food Supplements, Medical plants, Rutin, Total phenolic, Total flavonoids
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
Samples | |
---|---|
Plant material | Latin name |
Elderberry, fruit | Sambucus nigra |
Cranberry, fruit | Vaccinum vitis-ideae |
Hawthorn, fruit | Crataegus monogyna |
Wild mint, leaves | Mentha piperita |
Lemon balm, leaves | Melissa officinalis |
Food supplements | Description |
Elderberry (1) | Elderberry, Syrup, containing extract of Sambucus nigra fruit |
Elderberry (2) | Elderberry, Syrup, containing standardized extract of Sambucus nigra fruit |
Cranberry | Cranberry, Capsules, containing dry extract of Vaccinum vitis-ideae fruits |
Lemon balm | Lemon balm, Tablets, containing 300 mg Melissa officinalis |
Mint, Hawthorn, Valeriana | Mint, Hawthorn, Valeriana, Tablets, containing dry standardized extract of Crataegus monogyna – 120 mg,Valeriana officinalis – 100 mg, Mentha piperita – 30 mg and Matricaria chamomilla – 80 mg |
Hawthorn | Hawthorn, Tablets, containing 325 mg Crataegus monogyna |
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 (PDA), auto-sampler, column oven, and in-line degaser. Chromatographic data were processed with Chromera HPLC PDA Data Software, version 4.1.1.6396.
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 (LOD) and limit of quantfication (LOQ) were determined from Signal to Noise ratio, using progressively lower concentration of analytes for a S/N ratio of approximately 3 and of approximately 10, respectively (Eurachem 2014).
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, LOD, LOQ of the HPLC method for quantitative evaluation of (+)-catechin, (-)-epicatechin and rutin.
Compound | Calibration curve | Correlation coefficient R2 (n = 3) | Linear range (μg/ml) | RT, min | RSD, % (n = 6) | LOQ (μg/ml) | LOD (μg/ml) |
---|---|---|---|---|---|---|---|
(+)-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, LOD, LOQ of the spectrophotometric methods for determination of Total phenolic and Total flavonoids.
Calibration curve | Correlation coefficient R2 (n = 3) | Linear range (μg/ml) | LOQ (μg/ml) | LOD (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 (RSD, n = 6). Furthermore the intra- and inter-day precision was determined of hole analytical procedure by analyzing a cranberry fruit sample Vaccinum vitis-ideae (RSD, n = 6) (see Table
The recovery was evaluated by adding measured amount of pure standards to a sample of Sambucus nigra (fruit) and Cranberry Food supplement, following the extraction procedure described above, by analyzing the fortified samples with HPLC and spectrophotometric methods. The recoveries were determined by subtracting the values obtained for the control matrix from those of the samples prepared with added standards. Each sample was analyzed three times. The calculated mean amount of recovery was reported in Table
Repeatability. Reproducibility and Accuracy of determination of (+)-catechin. (-)-epicatechin and rutin and of Total phenolic and Total flavonoids.
RSD%1 | RSD%1 | RSD%2 | RSD%2 | Accuracy | ||
---|---|---|---|---|---|---|
Inter-Day (n = 6) | Intra-Day (n = 6) | Inter-Day (n = 6) | Intra-Day (n = 6) | (Analytical Recovery. %) (n = 3) | ||
HPLC Determination | ||||||
(+)-Catechin | 2.59 | 2.72 | 3.56 | 4.75 | Sambucus nigra. Fruit | 99.53 |
Cranberry. Supplement | 97.41 | |||||
(-)-Epicatechin | 2.00 | 2.35 | 1.94 | 3.53 | Sambucus nigra. Fruit | 92.46 |
Cranberry. Supplement | 93.57 | |||||
Rutin | 2.17 | 1.39 | 1.24 | 2.28 | Sambucus nigra | 96.02 |
Cranberry. Supplement | 95.97 | |||||
Total phenolic | 1.98 | 3.06 | 2.04 | 3.78 | Sambucus nigra. Fruit | 93.15 |
Cranberry. Supplement | 95.34 | |||||
Total flavonoids | 1.51 | 2.72 | 2.40 | 4.12 | Sambucus nigra. Fruit | 96.57 |
Cranberry. Supplement | 97.13 |
The results of HPLC analysis enabled the identification of (+)-catechin, (-)-epicatechin, and rutin within 12 min. The HPLC chromatograms of standard solutions and Sambucus nigra fruit and food supplement are presented in Figures
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.
The limits of quantification were 0.4 µg/ml for (+)-catechin and (-)-epicatechin, and 0.07 µg/ml for rutin (Table
Plant material | Total phenolic | Total flavonoids | (+)-Catechin | (-)-Epicatechin | Rutin |
---|---|---|---|---|---|
mg GAE/g | mg RE/g | µg/g | µg/g | µg/g | |
Sambucus nigra (fruit) | 16.88 | 16.24 | 149.6 | – | 2818.7 |
Vaccinum vitis-ideae (fruit) | 18.42 | 17.41 | 260.1 | 504.51 | 25.9 |
Crataegus monogyna (fruit) | 12.49 | 7.08 | – | 363.2 | 36.8 |
Mentha piperita (leaves) | 67.38 | 54.59 | – | – | 7332.5 |
Melissa officinalis (leaves) | 65.17 | 57.76 | – | – | 402.5 |
Food Supplements | |||||
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. Valeriana | 21.54 | 8.10 | 266.5 | 357.6 | 1214.7 |
Hawthorn | 28.76 | 9.95 | – | 618.1 | 26.2 |
(-)- lower than LOQ |
The results for limit of determination for Total phenolic and Total flavonoids measurements are 0.21 µg GAE/ml and 0.47 µg RE/ml (Table
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 Mentha piperita and Melissa officinalis have the highest values of Total phenolic and Total flavonoids (67.38 and 65.17 mg GAE/g; 54.59 and 57.76 mg RE/g, respectively. The lowest polyphenolic content was measured in Crataegus monogyna fruit – 12.49 mg GAE/g and 7.08 mg RE/g. Rutin was detected in all samples analysed, reaching the hinges level in Mentha piperita (7332.5 µg/g), followed by fruits of Sambucus nigra fruits (2818.7 µg/g). (+)-Catechin was detected only in Sambucus nigra and Vaccinum vitis-ideae fruit samples, while (-)-Epicatechin in Vaccinum vitis-ideae and Crataegus monogyna.
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 Sambucus nigra fruit. For instance Total phenolics content in Elderberry food supplement (1) is 5.16 mg GAE/g. while in Elderberry (2) was 20.76 mg GAE/g. The same tendency was observed for Total flavonoids and for rutin values. The highest phenolics content was measured in Lamon balm food supplement – 32.30 mg GAE/g. but this value is about two times lower than in Melissa officinalis leaves. The highest amount of Total flavonoids was found in Cranberry fruit supplement – 20.25 mg RE/g. It is interesting to notice that Mint, Hawthorn, Valeriana food supplement contains all individual flavonoids studied. Since (+)-catechin was not found in Mentha piperita or Crataegus monogyna samples, it is possible to come from other constituents of the Tablets form – Valeriana officinalis or Matricaria chamomilla. This food supplement formulation is also the richest source of rutin – 1214.7 µg/g.
Our results for Total phenolic and Total flavonoids in Lemon balm (Melissa officinalis) and mint (Mentha piperita) are higher than reported from
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 Mentha piperita leaves have a very high content of Total phenolic (67.38 mg GAE/g), Total flavonoids (54.59 RE/g) and of rutin (7.33 mg/g). In general, the food supplements studied have lower polyphenols values, in comparison with the selected medical herbs and fruits. The results of the study will be a practical contribution to a more complete knowledge and characterization of the polyphenolic composition in Bulgarian medicinal plants.
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.