|Year : 2018 | Volume
| Issue : 3 | Page : 66-72
Modeling of solvent effects on phytocompounds' extraction from glycyrrhizae radix
Nikolay Nikolaevich Boyko1, Dmitriy Pisarev1, Elena Zhilyakova1, Oleg Novikov1, Victoria Kuznietsova2, Natalia Sushchuk3
1 Scientific and Educational Centre “Pharmacy,” Belgorod State University, Belgorod, Russia
2 Department of Chemistry of Natural Compounds, National University of Pharmacy, Kharkiv, Ukraine
3 Department of Technology of Drugs, Odessa National Medical University, Odessa, Ukraine
|Date of Web Publication||28-Sep-2018|
Prof. Nikolay Nikolaevich Boyko
Scientific and Educational Centre “Pharmacy”, Belgorod State University, 85, Pobedy St., Belgorod, 308015
Source of Support: None, Conflict of Interest: None
The aim of the article was to study and model solvent effects on phytocompounds' extraction from Glycyrrhizae radix for substantiation of rational choice of the extractant in the technology of drugs obtained from this type of plant raw material. The process of extraction was carried out by simple maceration for 24 h at temperature 25°C ± 1°C and extractant/plant raw material ratio 5:1 (v/w). The content of hydro-ethanolic extracts based on ethanol solutions with concentration of 22, 41, 50, 71, 82, and 96% ± 1% v/v and some other solvents was studied by reversed phase high-performance liquid chromatography. The optimal range of solvent concentration for simultaneous extraction of chalcones and glycyrrhizic acid derivatives from Glycyrrhizae radix was determined. It was found that dielectric constant of the solvent plays a key role in the distribution process of phytocompounds between the phases; there is a certain range of dielectric constant values of the solvent-water solution, within which maximum phytocompound concentration in the extract can be observed; the dependency between phytocompound concentration in the extract and dielectric constant of the solvent-water solution can be described by equation lnC = a + b/ε+d/ε2.
Keywords: Glycyram, high-performance liquid chromatography analysis, licorice, licuroside, modeling
|How to cite this article:|
Boyko NN, Pisarev D, Zhilyakova E, Novikov O, Kuznietsova V, Sushchuk N. Modeling of solvent effects on phytocompounds' extraction from glycyrrhizae radix. J Adv Pharm Technol Res 2018;9:66-72
|How to cite this URL:|
Boyko NN, Pisarev D, Zhilyakova E, Novikov O, Kuznietsova V, Sushchuk N. Modeling of solvent effects on phytocompounds' extraction from glycyrrhizae radix. J Adv Pharm Technol Res [serial online] 2018 [cited 2020 Sep 19];9:66-72. Available from: http://www.japtr.org/text.asp?2018/9/3/66/242393
| Introduction|| |
Many plants from the Fabaceae family play an important role in human life due to their wide use in agriculture and medicine to obtain foods and drugs. A special place in this family belongs to plants of the Glycyrrhiza genus. Some plants of this genus accumulate polysaccharides, pectin substances, triterpene saponosides (in general as potassium and calcium salts of glycyrrhizic acid), tannins, as well as phenolic compounds (flavonoids, chalcones, coumarins, phenolcarbonic acids, etc.), organic acids, sugars, and some other compounds in their underground organs.,,,,
The main biologically active substances from Glycyrrhizae radix are triterpene saponosides (glycyrrhizic acid), flavonoids, and chalcones.,, These substances are responsible for some useful pharmacological effects, such as expectorative, anti-inflammatory, anti-allergic, healing, spasmolytic, antiviral, antibacterial.,,,,
It should be noted that many scientific works all over the world are dedicated to the phytochemical analysis of different extracts from Glycyrrhizae radix.,, However, the authors have found no systemic studies on the dependency between the qualitative and quantitative content of extracts from Glycyrrhizae radix and physical properties of the solvent, which is an important step for substantiation and development of a rational technology of drugs from this type of plant raw material.
The aim of the article was to study modeling of solvent effects on phytocompounds' extraction from Glycyrrhizae radix for substantiation of rational choice of the extractant in the technology of drugs obtained from this type of plant raw material.
| Materials and Methods|| |
Plant raw material
For study purposes used plant raw material “Glycyrrhiza radices” by privately held corporation “Liktravy,” Zhitomir, Ukraine, lot No. 50914, expiry date X/2019, and by privately held corporation “Krasnogorskleksredstva,” Krasnogorsk, Russia, lot No. 30417, expiry date V/2020.
Preparation of extracts
Before extraction, the plant raw material was grinded to particle size of 0.1–0.5 mm. The process of extraction was carried out by simple maceration for 24 h at temperature 25°C ± 1°C and extractant/plant raw material ratio 5:1 (v/w). For this purpose used hydro-ethanolic solutions with ethanol concentration of 22%, 41%, 50%, 71%, 82%, and 96% ± 1% v/v, determined with an alcoholmeter and ethanol 55% wt., 2-propanol 55% wt., acetone 55% wt., and methanol 80% ± 1% wt., determined by density.
Chemicals and reagents
For the preparation of hydro-ethanolic solutions used distilled water and ethanol for pharmaceutical and medicinal purposes 96% v/v. For the preparation of 2-propanol 55% wt., acetone 55% wt., and methanol 80% wt. solutions used analytically pure solvents. Qualitative analyses of compounds were carried out by reference substances of licuroside and monoammonium glycyrrhizate (glycyram) (Ukrainian State Pharmacopoeia, Ukraine), and by their ultraviolet-spectra and retention times in the high-performance liquid chromatography (HPLC) fingerprint.
High-performance liquid chromatography analysis
After maceration, the extracts were decanted, centrifuged at 13,000 rpm for 5 min, and analyzed by reversed phase HPLC. A reversed phase HPLC analysis was carried out on a chromatograph of “Agilent Technologies 1200 Infinity” type, the USA. The process of a reversed phase HPLC analysis was carried out under the following conditions: mobile phase (a): 1% water solution of formic acid, (b): ethanol 96% v/v in linear gradient elution; chromatographic column: Supelco Ascentis express C18, sized 2.7 μm × 100 mm × 4.6 mm; mobile phase velocity: 0.5 ml/min; temperature of the chromatographic column: +35°C; sample volume: 1 μl.
Control of chromatographic system suitability
The chromatographic system can be considered suitable if the following conditions are reached: the peak's theoretical plates (N) has to be not <1000; the peak's asymmetry coefficient (T) has to be <2.0; the peak's separation coefficient (RS) not <1.5; and relative standard deviation (RSD) of peak's area RSD has to be <2.0.
All the experiments were repeated four times. Statistical calculations were carried out using MS Excel 2010.
For mathematical modeling of the influence of solvent's dielectric constant on phytocompounds' equilibrium concentration in the extract, the authors used theoretical fundamentals of physical chemistry related to Van-der-Waals forces and Gibbs energy.
Herewith, Gibbs energy is related to the equilibrium constant of the phytocompound at a first approximation by the equation, (1) and energy of Van-der-Waals forces can be described by equations (2)– (6), all of which are related to the dielectric constant of the solvent:
where ΔG is Gibbs energy, J/mol; R is gas constant, J/(K·mol); T is absolute temperature, K; K is equilibrium constant; C is equilibrium concentration of the phytocompound in the extract, mol/l; ΔGsolid is cohesive energy of biologically active substances and plant raw material matrix molecules, J/mol; ΔGsolv is interaction energy of phytocompound and solvent molecules, J/mol; ΔGunpred is unpredicted energy processes, J/mol; NA is Avogadro number, mol − 1; π is mathematical constant; ε0 is electrical constant, F/m; ε1εx are dielectric constants of the plant raw material matrix and solvent; μ1μ2μ3 are dipole moments of molecules for plant raw material matrix, phytocompound, and solvent, respectively, C·m; α1α2α3 are polarizability of molecules for plant raw material matrix, phytocompound, and solvent, respectively, m3; I1I2I3 are ionization energy of molecules for plant raw material matrix, phytocompound, and solvent, respectively, J; e2,z2 are phytocompound's valency and charge; r is the distance between the molecules, m; and φ1 is the volume part of plant raw material matrix.
Equations (3) and (4) can be used for polar molecules, and equations (5) and (6) for ions. After substitution of equations (1), (3), and (4) into (2), we can obtain final equation (8) that represents the dependency between polar phytocompound concentration and dielectric constant of the solvent:
Equation (8) is relatively difficult for mathematical manipulation, and to simplify it, the authors supposed that relations εx2/εy2 and εx/εy are equal to the constant. In this case, the dependency will be defined in general by the balance of energy coefficients (D + E)-const·B and (G-const·A) and can predict both, extremum and plateau, in a certain range of solvent dielectric constant values.
| Results and Discussion|| |
[Figure 1] presents HPLC fingerprint chromatogram at 268.4 nm for the extract from Glycyrrhizae radix of “liktravy” based on hydro-ethanol extractant with ethanol concentration of 71% v/v. Extracts obtained with ethanol 22%, 41%, 50%, 82%, and 96% v/v had the same type of chromatograms with differences in the peak amplitude/area of the main components. It should be noted that glycyrrhizic acid derivatives were practically not detected in ethanol solution 96% v/v, while these compounds were detected in high quantity in more diluted solutions of ethanol, starting from 82% v/v and below.
|Figure 1: High performance liquid chromatography fingerprint of the extract based on 71% v/v ethanol at 268.4 nm with UV-spectra of the main compounds: I and II are flavonoids, III and IV are chalcones, V and VI are glycyrrhizic acid derivatives|
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As it can be seen from [Figure 1], dominative compounds in the extract based on ethanol solution 71% v/v from Glycyrrhizae radix are flavonoids (Compounds I and II), chalcones (Compounds III and IV), and glycyrrhizic acid derivatives (Compounds V and VI).
The data obtained correlate well with other works., Using reference standards, we identified three compounds. Compound IV is chalcone licuroside, and glycyrrhizic acid derivatives V and VI are constituents of Glycyram. Chromatographic system suitability parameters for these compounds are presented in [Table 1].
|Table 1: Chromatographic system suitability parameters for licuroside ana glycyram compounds|
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As it can be seen from [Table 1] all parameters for compounds in this chromatography system are satisfy the conditions and these compounds can be determined by HPLC analysis in our conditions. To evaluate the quantitative content of licuroside and glycyram in extracts, a HPLC analysis method was developed using absolute calibration. The standard solutions of Licuroside (from 9.00 to 0.103 mmol/l) and Glycyram (from 21.4 to 0.101 mmol/l) were analysis under the given above conditions, and the absolute calibration linear equations were obtained for sum of glycyrrhizic acid derivatives, it is C = (2.11 ± 0.06)·10 − 6·S, (mol/l), with determination coefficient equal to R2 =0.999 and for Licuroside, it is C = (6.10 ± 0.18)·10 − 7·S, (mol/L), with determination coefficient equal to R2 =0.999.
[Table 2] presents peak area values for the six compounds that dominate in the extracts.
|Table 2: Peak area and concentration values for compounds identified in the extracts from Glycyrrhizae radix of “Liktravy”, obtained from ethanol-water solutions with different concentrations|
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As it can be seen from the data in [Table 2], maximum peak areas for flavonoids (Compounds I and II) are observed for ethanol solution 50% v/v. Chalcones (Compounds III and IV) have maximum content in ethanol solution with concentration of 71% v/v. Glycyrrhizic acid derivatives (Compounds V and VI) have maximum peak area values in ethanol solution with concentration 50% v/v, but these values differ insignificantly from those in ethanol solution with concentration 41 and 71% v/v.
It should be noted that ethanol solution with concentration 96% v/v accumulated approximately 1.8-fold less flavonoids, 2.3-fold less chalcones, and 115-fold less glycyrrhizic acid in comparison with ethanol solution 82% v/v.
The fact of phenolic compounds' selective extraction into ethanol solution with concentration 96% v/v provides us with a tool for development of a step by step treatment package technology for Glycyrrhizae radix processing and separate production of extracts rich in phenolic compounds (flavonoids and chalcones) and glycyrrhizic acid derivatives.
From the data in [Table 2], we can also see that for extraction of all kinds of phytocompounds, the optimal range of ethanol concentration falls within the range of 40%–70% v/v. These results reveal easonability of development technology to obtain extracts (sicca, fluida and spissa) or a tincture from Glycyrrhizae radix in the territory of the Russian Federation and Ukraine using ethanol solution 55% ±15% v/v. In this case, an extract or a tincture will have maximum content of not only phenolic compounds but glycyrrhizic acid derivatives, too. In addition, we will see significant energy reduction at the stage of evaporation in comparison with water extracts.
It is interesting to note that European pharmacopeia contains a monograph describing licorice ethanolic liquid extract, standardized. According to requirements of this monograph, the extract is obtained from the Licorice root by a suitable method using ethanol solution of 70% v/v. Furthermore, the extract should contain glycyrrhizic acid from 3 to 5% w/w, and ethanol content in the extract should be within the range of 52–65% v/v, which almost completely agrees with our results.
To model dependence between phytocompound concentration and solvent dielectric constant according to equation (8), the authors used peak area values presented in [Table 2], the absolute calibration linear equations and reference data about the dielectric constant for ethanol-water solutions presented in work.
The relationship between the natural logarithm of concentration (lnC) of Licuroside and glycyrrhizic acid derivatives in the extracts and dielectric constant of ethanol-water solutions are presented in [Figure 2]. Values and their confidence interval (mean ± standard error of the mean) are calculated with repeat counts n = 4 and significance level P =0.95.
|Figure 2: Dependency between phytocompounds' natural logarithm of concentration in extracts and dielectric constant of ethanol-water solutions. (1) Glycyram (sum of glycyrrhizic acid derivatives, compound V and VI); (2) chalcone (Licuroside), compound IV|
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As it can be seen from [Figure 2], the relationship between the natural logarithm of glycyrrhizic acid derivatives (equation 1) and Licuroside (equation 2) concentration and ethanol-water dielectric constant has good approximation by equation (8) with determination coefficient R2 =0.977.
The equations obtained predict the maximum content of glycyrrhizic acid derivative accumulation in solvent-water solutions with the range of dielectric constant values of 48 ± 5 units, and for licuroside 40 ± 4 units.
Based on these data, it is reasonable to expect that solvent-water solution should have a range of dielectric constant values of 44 ± 4 units to reach simultaneously maximum concentration of these compounds in the extract.
To confirm this statement, an additional experiment was carried out using water solutions of ethanol 55% wt., 2-propanol 55% wt., acetone 55% wt., and methanol 80% wt. by virtue of the fact that these solutions have dielectric constant values within the range of 44 ± 2 units. The results obtained are presented in [Table 3].
|Table 3: Peak area and concentration values for compounds identified in extracts from Glycyrrhizae radix of “Krasnogorskleksredstva”, obtained from ethanol-water solutions with different concentrations|
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As it can be seen from the data of [Table 3], peak area values for licuroside in the extracts with 2-propanol 55% wt., acetone 55% wt., ethanol 55% wt. are comparable to each other (maximum deviation is 8%), but for methanol 80% wt., this value has a stronger deviation (equal to 22%).
For glycyrrhizic acid derivatives, the sum of their areas is comparable only for 2-propanol 55% wt., and ethanol 55% wt., for acetone 55% wt. this deviation is equal to 20%, and for methanol 80% wt., it is 48%.
Thus, it can be concluded that the most suitable solvents for simultaneous extraction of both, chalcones and glycyrrhizic acid derivatives, are water solutions of 2-propanol 55% wt., and ethanol 55% wt. Furthermore, acetone and methanol solutions rank below, by 20% and 48%, respectively.
The data obtained reveal a good correlation between the experiment and theory, but further studies are necessary to investigate and explain the abnormal behavior of methanol solution.
In addition, one interesting fact has been also found. For construction and analysis of phytocompound's content dependency in the extract on ethanol-water solution's dielectric constant, it is not compulsory to use absolute values of the lnC, but the logarithm of the ratio of peak area value to a maximum value (lnS/Smax) can be used. In this case, we obtain correlation equations almost identical to those presented in [Figure 2]. For example, for Glycyram we get: lnS/Smax= −7.9 + 795/ε-19004/ε2, with R2 = 0.977, and for Licuroside: lnS/Smax= −3.3 + 263/ε-5293/ε2, with R2 = 0.977. As it can be seen, the constants at the dielectric constant are identical (within the error) and different for the absolute term in the equation, which, however, does not interfere calculations of the optimal value of the dielectric constant. This fact discovered allows using the results of HPLC analysis without necessity to buy and use of the standard compound, which significantly simplifies data acquisition and their manipulation in studies of the kind.
Thus, according to this study, the following general conclusions can be made:first, solvent's dielectric constant plays a key role in the distribution process of phytocompounds between the phases; second, there is a certain range of dielectric constant values of the solvent-water solution, within which maximum phytocompound concentration in the extract can be observed; third, at a first approximation, the dependency between phytocompound concentration in the extract and dielectric constant of the solvent-water solution can be described by equation lnC = a + b/ε+d/ε2.
| Conclusion|| |
Solvent effects on phytocompounds extraction from Glycyrrhizae radix have been studied and modeled for substantiation of rational choice of the extractant in the technology of drugs obtained from this type of plant raw material. It is shown that the solvent's dielectric constant plays a key role in the distribution process of phytocompounds between the phases; there is a certain range of dielectric constant values of the solvent-water solution, within which maximum phytocompound concentration in the extract can be observed; the dependency between phytocompound concentration in the extract and dielectric constant of the solvent-water solution can be described by equation lnC = a + b/ε+d/ε2. It is determined that ethanol solution 96% v/v extracts predominantly flavonoids and chalcones, but does not extract glycyrrhizic acid derivatives, which provides a reason for the development of a step by step treatment package technology for this type of plant raw material. An optimal range of ethanol concentration in hydro-ethanolic solutions for simultaneous extraction of phenolic compounds and glycyrrhizic acid derivatives has been found, and it falls within the range of 62.5% ±7.5% v/v. Other the most suitable solvents for simultaneous extraction of both, phenolic compounds and glycyrrhizic acid derivatives, are water solutions of 2-propanol, and acetone (55% wt.).
Authors express their highest esteem and thanks to Doct. Chem. Sc., Prof. Vasiliy Ivanovich Litvinenko, the Head of the Laboratory of Chemistry and Technology of Phytochemical Drugs of State Enterprise “State Scientific Center of Drugs and Medicinal Products”, Kharkiv, Ukraine, for his help with reference substances.
Financial support and sponsorship
The results were obtained under state grant No. 12.6429.2017/BCh “Complex research of plant-origin objects in the process of creating targeted dosage forms for proctology.”
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]