Corresponding author: Liliia M. Horiacha (
The qualitative composition and quantitative content of phenolic compounds were studied by the HPLC method. The performed experiment revealed presence of hydroxycinnamic acids, flavonoids, isoflavonoids and coumarins in tested herb.
The content of phenolic compounds was the highest in
Quantitatively neochlorogenic acid dominated in flowers, chlorogenic acid in fruits, whereas
The obtained results will be useful in the development of quality control methods for
Pharmacological researchers in various countries confirmed anti-bacterial, anti-fungal, anthelmintic, anti-inflammatory, diuretic activity (
Nigerian scientists established anti-malaria activity of
The object of the present work was the study of phenolic compounds in
The identification of the chromatographic peaks was achieved by comparing the retention times and spectral characteristics (λ = 200–600 nm)of the eluting peaks with those of the reference compounds (Table
The following validation characteristics of method were evaluated as specificity, reproducibility of results (accuracy), limits of detection and quantitative evaluation, linearity (Tables
The method specificity was confirmed by coincidence between peak retention time of identified phenolic compounds and the corresponding peak retention time in standard samples and divisibility of substance peaks in chromatogram.
Retention time, detected at wavelength and UV spectrum of identified phenolic compounds.
Compound | Retention time, min | Detected at wavelength λ, nm | UV spectrum |
---|---|---|---|
Neochlorogenic acid | 9.86 ± 0.20 | 320 |
|
Chlorogenic acid | 11.96 ±0.30 | 320 |
|
Caffeic acid | 14.18 ± 0.30 | 320 |
|
Rutin | 21.48 ± 0.50 | 353 |
|
Ferulic acid | 23.16 ± 0.50 | 320 |
|
Hyperoside | 23.89 ± 0.30 | 350 |
|
Isoquercitrin | 24.83 ± 0.20 | 350 |
|
Avicularin | 30.94 ± 0.65 | 350 |
|
Coumarin | 33.77 ± 0.30 | 275 |
|
Ononin | 39,52 ± 0.20 | 350 |
|
Linearity, LOD, LOQ of the HPLC method for the quantitative evaluation of phenolic compounds.
Compound | Calibration curve | Correlation coefficient R2 ( |
Linear range (μg/mL) | RSD, % | LODb (μg/mL) | LOQc (μg/mL) |
---|---|---|---|---|---|---|
Neochlorogenic acid | f(x) = 30834.6*x-613.100 | 0.9999 | 0.38–48.00 | 1.18 | 0.03 | 0.08 |
Chlorogenic acid | f(x) = 29930.2*x-538.361 | 0.9999 | 0.36–46.00 | 1.29 | 0.02 | 0.07 |
Caffeic acid | f(x) = 57646.8*x-3853.48 | 0.9999 | 0.72–91.92 | 1.56 | 0.02 | 0.06 |
Ferulic acid | f(x) = 54955.4*x-638.345 | 0.9999 | 0.44–56.50 | 1.60 | 0.03 | 0.08 |
Rutin | f(x) = 16072.5*x+1499.73 | 0.9999 | 0.16–20.24 | 1.07 | 0.09 | 0.29 |
Hyperoside | f(x) = 22498.4*x+2508.57 | 0.9999 | 0.21–27.04 | 1.19 | 0.07 | 0.23 |
Isoquercitrin | f(x) = 24139.7*x+3904.44 | 0.9999 | 0.35–44.56 | 1.02 | 0.07 | 0.22 |
Ononin | f(x) = 37456.2*x+5132.42 | 0.9998 | 0.21–26.24 | 1.29 | 0.05 | 0.15 |
Avicularin | f(x) = 16508.5*x-158.335 | 0.9999 | 0.16–10.39 | 1.35 | 0.004 | 0.01 |
Coumarin | f(x) = 34481.8*x+42631.1 | 0.9999 | 1.85–237.50 | 0.81 | 0.14 | 0.42 |
Precision of the HPLC method for the quantitative evaluation of phenolic compounds.
Compound | Concentrate (µg/mL) | Deviation (%) | Intra-Day (n = 3) | Inter-Day (n=3) | ||
---|---|---|---|---|---|---|
RSD (%) | Accuracy (%) | RSD (%) | Accuracy (%) | |||
Neochlorogenic acid | 6.05 | 100.17 | 0.87 | 100.35 | 0.88 | 101.16 |
24.12 | 98.58 | 1.25 | 99.17 | 0.97 | 102.37 | |
48.43 | 100.14 | 1.22 | 100.25 | 0.71 | 101.27 | |
Chlorogenic acid | 5.75 | 100.69 | 1.31 | 101.12 | 0.38 | 98.40 |
23.20 | 99.58 | 0.42 | 99.08 | 0.73 | 99.43 | |
46.58 | 101.91 | 0.96 | 100.27 | 0.48 | 98.24 | |
Caffeic acid | 11.49 | 100.01 | 1.05 | 102.02 | 0.52 | 98.49 |
45.96 | 99.39 | 1.08 | 98.78 | 0.67 | 99.73 | |
91.92 | 100.17 | 0.64 | 100.35 | 0.95 | 98.17 | |
Ferulic acid | 7.06 | 99.11 | 0.68 | 100.22 | 0.90 | 98.29 |
28.25 | 99.60 | 0.93 | 98.20 | 0.29 | 99.31 | |
56.50 | 100.12 | 1.22 | 100.24 | 0.46 | 98.28 | |
Rutin | 2.53 | 100.18 | 1.26 | 100.35 | 0.62 | 100.15 |
10.12 | 100.65 | 1.29 | 101.12 | 0.80 | 99.21 | |
20.24 | 99.78 | 0.76 | 99.56 | 1.14 | 100.94 | |
Hiperoside | 3.433 | 100.78 | 0.99 | 101.06 | 0.86 | 98.95 |
13.72 | 100.72 | 0.50 | 101.04 | 0.70 | 99.07 | |
26.92 | 99.77 | 0.42 | 99.53 | 0.80 | 100.97 | |
Isoquercitrin | 5.57 | 100.13 | 0.86 | 100.26 | 0.41 | 100.23 |
22.28 | 99.64 | 1.12 | 101.27 | 0.98 | 99.24 | |
44.56 | 97.79 | 0.80 | 99.58 | 0.91 | 100.92 | |
Ononin | 3.28 | 100.56 | 0.81 | 100.12 | 0.32 | 98.39 |
13.12 | 100.72 | 0.69 | 100.44 | 0.75 | 99.08 | |
26.24 | 99.75 | 0.84 | 99.50 | 0.82 | 100.00 | |
Avicularin | 0.32 | 100.15 | 1.02 | 98.75 | 1.06 | 100.63 |
1.29 | 99.59 | 0.78 | 101.78 | 0.97 | 99.12 | |
5.19 | 99.41 | 0.94 | 100.10 | 0.71 | 99.95 | |
Coumarin | 1.85 | 99.82 | 0.79 | 100.12 | 0.98 | 100.02 |
7.42 | 99.57 | 0.86 | 99.34 | 1.07 | 99.87 | |
29.68 | 100.11 | 0.90 | 98.99 | 1.10 | 99.32 |
The limit of detection (LOD) may be expressed as: DL = 3.3 *σ/S, where σ = the standard deviation of the response, S = the slope of the calibration curve. The limit of quantification (LOQ) may be expressed as DL = 3.3 *σ/S, where σ = the standard deviation of the response, S = the slope of the calibration curve. The determined LOQ was within the range from 14.00 ng/mL to 420.00 ng/mL. The obtained results confirmed method eligibility for quantitative evaluation of phenolic compounds.
The correlation coefficient (
The total content of phenolic compounds was calculated by summing the identified components.
The results of experiment enabled the identification of hydroxycinnamic acids, flavonoids, isoflavonoids and coumarins in tested
The HPLC chromatogram of
Nine phenolic compounds were detected in
The quantitative content of phenolic compounds as determined by the HPLC method in
The content of phenolic compounds in
Сompound | Flowers | Roots | Fruits | Herb | ||||
---|---|---|---|---|---|---|---|---|
Retention time, min | Quantitative content, µg/mg | Retention time, min | Quantitative content, µg/mg | Retention time, min | Quantitative content, µg/mg | Retention time, min | Quantitative content, µg/mg | |
Neochlorogenic acid | 9.92 | 2060.10 ± 41.20 | – | – | – | – | 9.94 | 69.87 ± 3.49 |
Chlorogenic acid | – | – | – | – | 10.98 | 51.73 ± 1.03 | 11.36 | 5.30 ± 0.11 |
Caffeic acid | 14.04 | 76.77 ± 1.54 | 13.88 | 4.40 ± 0.09 | 14.26 | 10.13 ± 0.20 | 14.05 | 8.60 ± 0.17 |
Rutin | 21.09 | 131.63 ± 2.63 | – | – | – | – | 21.23 | 110.33 ± 2.21 |
Ferulic acid | 22.73 | 79.37 ± 1.59 | 23.67 | 4.57 ± 0.09 | – | – | 22.80 | 12.93 ± 0.26 |
Hyperoside | 23.66 | 292.67 ± 5.85 | – | – | 23.67 | 4.37 ± 0.09 | 23.77 | 93.20 ± 1.86 |
Isoquercitrine | 24.89 | 3.20 ± 0.06 | – | – | – | – | 24.87 | 1.00 ± 0.02 |
Avicularin | 31.55 | 322.30 ± 6.45 | – | – | – | – | – | – |
Coumarin | 33.55 | 8.96 ± 0.18 | – | – | – | – | 33.62 | 3.02 ± 0.06 |
Ononin | 39.58 | 2.41 ± 0.05 | – | – | 39.41 | 1.69 ± 0.03 | – | – |
The HPLC chromatogram of phenolic compounds in
The HPLC chromatogram of phenolic compounds in
The HPLC chromatogram of phenolic compounds in
The HPLC chromatogram of phenolic compounds in
The highest content of phenolic compounds was found in
In
The lowest content of phenolic compounds among tested parts of herb was in roots – 12.44 µg/mg. The amounts of ferulic and caffeic acids in these was almost identical (4.57 µg/mg and 4.40 µg/mg respectively).
Our experiment enabled the identification of flavonoids, isoflavonoids, hydroxycinnamic acids and coumarins in
The obtained results will be useful in the development of quality control methods for