Corresponding author: Liliia Budniak ( stoyko_li@tdmu.edu.ua ) Academic editor: Plamen Peikov
© 2020 Liliia Budniak, Marjana Vasenda, Svitlana Marchyshyn, Khrystyna Kurylo.
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:
Budniak L, Vasenda M, Marchyshyn S, Kurylo K (2020) Determination of the optimum extraction regime of reducing compounds and flavonoids of Primula denticulata Smith leaves by a dispersion analysis. Pharmacia 67(4): 373-378. https://doi.org/10.3897/pharmacia.67.e54170
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Herbal medicines are widely used in the complex treatment of various diseases. Therefore, theoretical and practical interest is the in-depth study of drumstick primrose (Primula denticulata Smith). The study aimed to determine the optimal extraction mode of flavonoids and reducing compounds of drumstick primrose leaves. The concentration of ethanol, the ratio of raw materials and extractant, and extraction method were studied by dispersion analysis. This allowed reducing the number of experiments from 64 to 16. To obtain the alcohol extract of drumstick primrose leaves with the highest content of reducing compounds and flavonoids, found that maceration is the optimal method of extraction, the ratio of raw materials to extractant should be 1 to 5 and 40% ethanol is the most appropriate extractant.
Primula denticulata Smith, dispersion analysis, flavonoids, reducing compounds
During many centuries, plants have been used not only as a source of nutrition but also in the struggle with diseases (
It is estimated that 80% of the world’s population living in the developing world relies on herbal medicinal products as a primary source of healthcare. A traditional medical practice that involves the use of herbs is viewed as an integral part of the culture in those communities (
Theoretical and practical interest leads to an in-depth study of drumstick primrose (Рrimula denticulata Smith).
Рrimula denticulata Smith, commonly known as drumstick primrose , belongs to family Primulaceae (
The plant contains various active ingredients (secondary plant metabolites) as alkaloids, phenolics, flavonoids, tannins, cardiac glycosides, terpenes, saponins, steroids, coumarins and carbohydrates (
The chemical profiling of Рrimula denticulata Smith showed the presence of 5-hydroxyflavone 5,8-dihydroxyflavone, 2’-hydroxyflavone, and 5,2’-dihydroxyflavone (
The HPLC determined the qualitative composition and quantitative content of glycosides of flavonols – rutin, isoquercitrin, hyperoside and aglycones: flavons – luteolin, apigenin; flavonols – kaempferol (
In the literature sources, there is evidence of the healing properties of Рrimula denticulata Smith.
Ethanolic extract of Рrimula denticulata Smith shows wound healing, antioxidant, antibacterial, antidiabetic activities (
In the world practice plant extracts are widely used as curative and preventive nutrition in the production of specialized food products (
The aim of the study was to determine the optimal extraction mode of flavonoids and reducing compounds of Рrimula denticulata Smith leaves.
Therefore, the influence of the extraction method, the nature of the extractant, the ratio of raw materials to extractant were studied. These factors have the greatest impact on the process of extraction of biologically active substances (BAS) from the studied raw materials.
During planning an experiment, mathematical methods were used both at the stage of processing the results, and the preliminary stage of experimentation, so-called stage of the experiment plan formation (
Leaves of the Primula denticulata Smith were collected in Western Ukraine, Tysmenetsk district, Ivano-Frankivsk region (49°01’18.2’’N, 24°40’34.4’’E), during a mass flowering period in 2018. The raw material was authenticated by prof. Svitlana Marchyshyn (TNMU, Ternopil, Ukraine). A voucher specimen no. 239 is kept in the Department of Pharmacognosy and Medical Botany, TNMU, Ternopil, Ukraine.
Spectrophotometer UV/VIS Lambda 25 (Perkin Elmer, USA) was used. A 1-cm quartz cell was used.
Rutin and pyrogallol were used as standards. Rutin (analytical grade 94% purity) and pyrogallol (analytical grade ≥98% purity) were obtained from Sigma-Aldrich. All other reagents were of the highest purity available.
For the planning of the experiment we used one of the dispersion analysis plans – 4×4 Latin square of the third order (
List of technological factors studied during extraction of Рrimula denticulata Smith.
Factor | Level of factor |
---|---|
A – the type of extractant | а1 – 20% ethanol |
а2 – 40% ethanol | |
а3 – 50% ethanol | |
а4 – 70% ethanol | |
В – the ratio of raw materials to extractant | b1 – 1 : 5 |
b2 – 1 : 8 | |
b3 – 1 : 10 | |
b4 – 1 : 12 | |
С – extraction method | c1 – maceration |
c2 – maceration with stirring | |
c3 – remaceration | |
c4 – ultrasonic extraction |
The matrix of experiment planning and research results are given in Table
The matrix of experiment planning and results of extraction of flavonoids and reducing compounds of Рrimula denticulata Smith.
Series No. | А | В | С | Content of flavonoids, mg/g | Content of reducing compounds, mg/g |
---|---|---|---|---|---|
1 | a1 | b1 | c1 | 0.93 | 0.84 |
2 | a1 | b2 | c2 | 0.26 | 0.74 |
3 | a1 | b3 | c4 | 0.22 | 0.71 |
4 | a1 | b4 | c3 | 0.14 | 0.63 |
5 | a2 | b1 | c2 | 0.99 | 1.08 |
6 | a2 | b2 | c1 | 0.51 | 0.98 |
7 | a2 | b3 | c3 | 0.37 | 0.80 |
8 | a2 | b4 | c4 | 0.32 | 0.91 |
9 | a3 | b1 | c3 | 0.68 | 0.96 |
10 | a3 | b2 | c4 | 0.45 | 0.85 |
11 | a3 | b3 | c1 | 0.54 | 0.87 |
12 | a3 | b4 | c2 | 0.48 | 0.80 |
13 | a4 | b1 | c4 | 0.57 | 0.81 |
14 | a4 | b2 | c3 | 0.73 | 0.91 |
15 | a4 | b3 | c2 | 0.82 | 0.88 |
16 | a4 | b4 | c1 | 0.72 | 0.82 |
The leaves of the Рrimula denticulata Smith were ground and mixed with extractant (20%, 40%, 50%, and 70% ethanol). Maceration, maceration with stirring, remaceration and ultrasonic extraction were used as methods of extraction.
During maceration and maceration with stirring, the raw material filled with the extractant was being infused for seven days. The resulting extracts were drained, the residue of plant raw materials was washed with an extractant. The extracts were then combined and filtered through a paper filter. The difference between these methods is in the periodic mixing of raw materials with the extractant.
During remaceration, the amount of extractant was divided into four portions and each portion was being infused with the raw material within 24 hours.
The device «Ultratone» with a frequency of ultrasonic waves 50 Hz was used as a source of ultrasound during ultrasonic extraction (
The evaluation criterium was the content of the sum of flavonoids and reducing compounds , the quantitative determination of which was carried out by the method of spectrophotometry.
The content of flavonoids was determined by this method.
Test solution. Aliquot of the obtained alcohol extract is placed into a 25 ml volumetric flask, added 10 ml of alcohol (70% (vol/vol)), 2.0 ml of 3% alcohol (70% (vol/vol)) solution of aluminum chloride, added alcohol (70% (vol/vol)) to the mark and it is mixed.
Compensatory solution. Aliqout of the obtained alcohol extract is placed into a 25 ml volumetric flask and added alcohol (70% (vol/vol)) to the mark and it is mixed.
Standard sample solution of rutin. 0.05 g (exact weight) of the standard sample of rutin is placed in a 100 ml volumetric flask, then 70 ml of alcohol (70% (vol/vol) are added, dissolved and added alcohol (70% (vol/vol)) to the mark and stirred.
Comparison solution. 1.0 ml of the standard sample solution of rutin is placed in a 25 ml volumetric flask, added 2.0 ml of 3% alcohol (70% (vol/vol)) aluminum chloride solution, and added alcohol (70% (vol/vol)) to the mark and stirred.
Compensatory solution. 1.0 ml of the standard sample solution of rutin is placed in a 25 ml volumetric flask and added alcohol (70% (vol/vol)) to the mark and stirred.
The optical density of the test solution and the comparison solution are measured 45 min after preparation at wavelength 408 nm relatively to the compensatory solutions for each one respectively.
The content of the sum of flavonoids in liquid extracts (X) in mg /g and in terms of rutin is calculated by the formula:
,
where: A – the optical density of the test solution;
A0 – the optical density of the comparison solution;
m0 – the mass of the standard sample of rutin, in grams;
ma – the mass of aliquot of the extract taken for analysis, in grams (
The content of reducing compounds was determined by spectrophotometric method (The State Pharmacopoeia of Ukraine 2015;
Initial solution. The aliquot of the obtained alcohol extracti is placed in a 25 ml volumetric flask and added waterto the mark, stirred and, if necessary, filtered.
Tested solution. 2.0 ml of the initial solution is placed in a 25 ml volumetric flask, 1.0 ml of phosphorus-molybdenum-tungsten reagent and 10.0 ml of water are added and added solution of 290 g/l sodium carbonate to the mark, stirred.
Standard solution. 50.0 mg of pyrogallol is placed in a 100 ml volumetric flask and added water to the mark, stirred. 5.0 ml of the obtained solution is placed in a 100 ml volumetric flask and added water to the mark, stirred.
Comparison solution. 2.0 ml of a standard solution of pyrogallol is placed in a 25 ml volumetric flask, 1.0 ml of phosphorus-molybdenum-tungsten reagent and 10.0 ml of water are added and added solution of 290 g/l sodium carbonate to the mark, mixed. 30 minutes later, the optical density of the tested solutions and the comparison solution are measured at wavelength 760 nm, using water as a compensatory solution.
The content of reducing compounds in the liquid extract (X) in mg/g and in terms of pyrogallol is calculated by the formula:
where: A – the optical density of the test solution;
A0 – the optical density of the comparison solution;
m0 – the mass of the standard sample of pyrogallol, in grams;
ma – the mass of aliquot of the extract taken for analysis, in grams (
The results were undergonethe dispersion analysis. The data were interpreted using the method of 4×4 Latin squares (Microsoft Office Excel, 2010), which allows us to conduct statistical processing of research results quickly.
Regression or dispersion analysis is used to establish the optimal mode of extraction of plant raw materials and obtain extract with the highest content of BAS. These analyses make it possible to reduce the number of experiments.
The regression analysis was used in development of optimal technology of alcohol extract Centaurium erythraea Rafn. with the highest BAS content. As a result of studies, ethanol concentration and the ratio of raw materials to extractant were determined which are 69% and 1 to 5 respectively (
Dispersion analysis was used to study the influence of technological parameters (extraction method, extractant concentration, degree of grinding of plant raw materials) on the extraction of BAS of walnut membranes. It is determined that the best method to obtain an extract with a high content of BAS is maceration, it is advisable to use 35% ethanol as an extractant, the degree of grinding of the raw material should be 0.5 mm (
For determination of the optimum extraction regime of flavonoids and reducing compounds of drumstick primrose leaves dispersion analysis method was also used.
In Figures
During extraction of reducing compounds (Figure
The influence of ratio of raw material to extractant on the flavonoid extraction (Figure
A similar result was obtained by the extraction of reducing compounds (Figure
The dependence of the degree of extraction of flavonoids and reducing compounds from Рrimula denticulata Smith leaves on the method of extraction is shown in Figures
The quantitative content of the studied substances of extracts that were obtained by maceration with stirring became slightly inferior. The smallest amount of flavonoids and reducing compounds is extracted by ultrasonic extraction.
The optimal extraction regime of reducing compounds and flavonoids was determined by the dispersion analysis. After analyzing the experimental data, it can be argued that the optimal extraction of flavonoids and reducing compounds of Рrimula denticulata Smith leaves, was reached when we used maceration as the extraction method, 40% ethanol as the most appropriate extractant and correlation of raw materials to extractant 1:5.