Study of distribution of biologically active substances from flowers of helichrysum arenarium between phases of the extraction system

Том 7, Выпуск 5, 2019 For citation: N.N. Boyko, E.T. Zhilyakova, A.Yu. Malyutina, D.K. Naplekov, N.N. Shestopalova, D.S. Martceva, O.O. Novikov, D.I. Pisarev, P.G. Mizina. Study of distribution of biologically active substances from flowers of helichrysum arenarium between phases of the extraction system. Pharmacy & Pharmacology. 2019;7(5): 271-278. DOI: 10.19163/2307-9266-2019-7-5-271-278 © Н.Н. Бойко, Е.Т. Жилякова, А.Ю. Малютина, Д.К. Наплеков, Н.Н. Шестопалова, Д.С. Марцева, О.О. Новиков, Д.И. Писарев, П.Г. Мизина, 2019 Для цитирования: Н.Н. Бойко, Е.Т. Жилякова, А.Ю. Малютина, Д.К. Наплеков, Н.Н. Шестопалова, Д.С. Марцева, О.О. Новиков, Д.И. Писарев, П.Г. Мизина. Изучение распределения биологически активных веществ из цветков бессмертника песчаного между фазами экстракционной системы. Фармация и фармакология. 2019;7(5): 271-278. DOI: 10.19163/2307-9266-2019-7-5-271-278 stUDY oF DIstRIBUtIon oF BIoLoGICALLY ACtIVe sUBstAnCes FRoM FLoWeRs oF HeLICHRYsUM ARenARIUM BetWeen PHAses oF tHe eXtRACtIon sYsteM

In our previous research [21], the authors justified the mechanism of dielectric constant impact on equilibrium concentration of isosalipurposide in the extractions. However, the obtained model neither explains nor describes the mechanism of BAS distribution in the extraction system when the equilibrium status is reached within it. That is why the studies aimed at the development of the equilibrium status of the extraction process are relevant. THE AIM of this study is to confirm the adequacy of the proposed hypothesis, which explains and quantitatively describes the distribution of BAS in the extraction system of Helichrysum arenarium flowers and the solvent using a regressive analysis in the theoretically predicted coordinates.

MATERIALS AND METHODS Raw materials and chemical reagents
For this study, milled plant raw material of Helichrysum arenarium was used, and it was bought at pharmacy Medical herbs Ltd., Kharkiv, Ukraine, lot number 530617, expiry date 07.2020 [22].
An aqueous solution of ethanol 80%±1%, was used as an extracting solution. Qualitative and quantitative analyses were carried out using RP HPLC with reference substances.
The main validation parameters of the analytical method and suitability of RP HPLC system for the assay of isosalipurposide, salipurposide, and chlorogenic acid, are shown in Table 1.

Methods of obtaining extracts
A precisely weighed amount of the raw material (1 g) was put into a hermetic flask, the required volume of the solvent was added, which additionally was weighted and put into a refrigerator/thermostat at the temperature of 4, 20, 40 and 60±1°С. The proportions of the raw material and the solvent at each temperature, were 1:5 (1:10), 1:15, 1:20, 1:40 w/v. The extraction mixture was decocted for 24 hours. After that, the extract was removed and its assay was performed using RP HPLC method. The mean value and the standard error of mean were calculated at the repeat count n=3 and the significance level P=0.95.

Analysis methods via RP HPLC
The analysis of the extractions was carried out using the chromatographic equipment of Agilent Technologies, "Agilent 1200 Infinity" series, manufactured in the USA. The detailed description is available in the following articles [21,23].

Theoretical part
In order to explain the mechanism and a quantitative description of BAS distribution between a solid phase of the medicinal plant raw material and a liquid phase of the solvent, the authors proposed the following hypothesis: the mechanism of equilibrium molecular distribution of BAS between two phases in the extraction system is explained and described by the classic Boltzmann distribution for discrete values of molecular energy (or quantum distribution according to Fermi and Dirac), equation (1). This hypothesis allows developing a mathematical model, which will describe the experimental data in the theoretically predicted coordinates, as shown by equations (2) and (3): where n is quantity of BAS in the solvent with energy equals ΔG, mol; n 0 is an overall quantity of BAS, mol; ΔG is the difference of Gibb's energy for BAS molecules in the extraction system, J; k is Boltzmann constant value, 1.38·10 -23 J/K; T is absolute Kelvin's temperature, K. (2) (3) where C is concentration of BAS in the extracting solution, g/ml; m 0 is an overall (initial) content of BAS in the raw material, g; V is the volume of the extracting solution, ml; М is the weight of the raw material in the extraction system, g; a is a constant that equals to the reversed value of overall BAS content in the raw material (М/m 0 ); b is a constant that equals to multiplicity of Henry's constant and (К Н ) and the reversed value of overall BAS content in the raw material (М/m 0 ), ml/g.
To determine the degree of adequacy of the proposed hypothesis, the authors used the regressive analysis of the experimental data in the theoretically predicted coordinates 1/С=f(V) and ln(b/a)=f(1/T). The obtained data were processed with preset for the data analysis in MS Excel 2010. DOI: 10.19163/2307-9266-2019-7-5-

RESULTS AND DISCUSSIONS
The experimental data and regressive linear equations of isosalipurposide, salipurposide, and chlorogenic acid concentration dependence on the volume of the extracting solution in the theoretically predicted coordinates are shown in Figures 1, 2, and 3.
As Figures 1, 2, and 3 show, the experimental points are well-approximated by regressive linear theoretically predicted coordinates 1/С=f(V). Wherein, the coefficient of determination of the regressive equations was R²≥0.998. It determines the functional dependence between the studied parameters and confirms the adequacy of the equation (2).
Thereafter, the obtained results were used for building up the regressive linear equations of dependence of Henry's constant for isosalipurposide, salipurposide and chlorogenic acid in the theoretically predicted coordinates. They are shown in Figure 4.
As Figure 4 shows, the dependence of Henry's constant values on the temperature is well-approximated for isosalipurposide, salipurposide, and chlorogenic acid by regressive linear equations in the theoretically predicted coordinates ln(b/a)=f(1/T). Wherein, the coefficient of the determination of the regressive equations was R²≥0.998. It proves the functional dependence between the studied parameters and confirms the adequacy of the equation (3). However, the obtained results have identified the additional constant value (g) in the equation (3), which was not predicted by the theory, hence, this requires to add this experimentally found constant value (g=ln(m p /100)) into the initial equation (1).
The constant values (ΔG, g and m p ) for BAS from Helichrysum arenarium flowers, found in accordance with the proposed theoretical equations (2) and (3) and also the experimental outcome, are summarized in Table 2.
As Table 2 shows, constant value ΔG, which expresses the energy of the BAS interphase distribution,

Figure 4 -Regressive equations of empiric constant values' (а, b) dependence on temperature in coordinates ln(b/a)=f(1/T) for BAS from Helichrysum flowers
is found at 5-20 kJ/mol level. It is well-complied with the values of the physical adsorption of the substances on the adsorbents [24]. Judging by this fact, BASes in the medicinal plant raw material are found to be in a bound or even adsorptive status, as it was discovered by M.S. Tsvet at the beginning of the XXth century [25].
The conclusive test of the proposed hypothesis was performed, comparing the experimentally obtained and theoretically calculated values of the overall (initial) content of BAS in the raw material (m 0 /M), which are shown in Table 3.
As Table 3 shows, the experimentally found and theoretically calculated values of the overall (initial) content of BAS in Helichrysum arenarium flowers (m 0 /M), do not differ from each other. This fact additionally confirms the adequacy of the equation (2).
Hence, the obtained experimental results are wellcomplied with theoretically developed mathematical models via equations (2) and (3). However, the experiment identified the necessity of adding constant (g) into the mathematical model, wherein equation (1) transforms to the following: (4) Hence, the proposed hypothesis concerning the mechanism of the interphase distribution of BAS in the extraction system, is explained and described by the classic Boltzmann distribution for discrete values of the molecular energy (or quantum distribution according to Fermi and Dirac), is not denied.
Thus, the proposed hypothesis and the developed mathematical model based on it, explain the mechanism of the BAS distribution in the extraction system between the phases; make it possible for us to find the constants required; forecast the equilibrium (limiting) concentration of BAS in the extract; and choose/calculate the optimal values of the volume and temperature of the extractant to achieve certain values of the exhaustive degree of the plant raw material for BAS.

CONCLUSION
A new hypothesis was proposed to explain and quantitatively describe the distribution of BAS in the extraction system of Helichrysum arenarium flowers and 80% ethanol. With this hypothesis, the mathematical models have been developed, their adequacy has been proved using a regressive analysis in the theoretically predicted coordinates. The constant values have been found, they are present in the mathematical model. The necessity of the addition of a new constant value has been identified experimentally. The obtained results do not deny that the mechanism of BAS distribution between the phases is explained and described by the classic Boltzmann distribution for discrete values of the molecular energy (or quantum distribution according to Fermi and Dirac).

ACKNOWLEDGMENT
For provision of reference substances, the authors sincerely express their acknowledgment to Vasiliy Ivanovich Litvinenko, Doctor of Sciences (Chemistry), the Head of the Laboratory of phytochemistry and technology of drug products at the government enterprise "State Scientific Center of Drugs and Medical Products, Kharkiv, Ukraine.

FINANCIAL SUPPORT
The results were obtained within the framework of the completion of state assignment No. 12.6429.2017/ BCh Consolidated studies of the objects of the natural origin to develop the row of targeted dosage forms for proctology.