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ФАРМАЦИЯ И ФАРМАКОЛОГИЯ
(с0
study of distribution of biologically active substances from flowers of helichrysum arenarium between phases of the extraction system
N.N. Boyko1, E.T. Zhilyakova1, A.Yu. Malyutina1, D.K. Naplekov1, N.N. Shestopalova1, D.S. Martceva1, O.O. Novikov2, D.I. Pisarev2, P.G. Mizina3
1 Laboratory of Drugs Technology, bDepartment of Pharmaceutical Technology, Belgorod National Research University
85, Pobeda Str., Belgorod, Russia 308015
2 Test Department "Drugs quality control center" of the Shared Research and Education Center Peoples' Friendship University of Russia, 8/2, Miklukho-Maklay Str., Moscow, Russia 117198
3 All-Russian Scientific Research Institute of Medicinal and Aromatic Plants 7, Grin Str., Moscow, Russia 117216
E-mail: boykoniknik@gmail.com
Received 6 September 2019 Review (1) 10 October 2019 Review (2) 15 October 2019 Accepted: 21 October 2019
The aim of this study is to confirm the adequacy of the proposed hypothesis, which explains and quantitatively describes the distribution of biologically active substances (BAS) within the extraction system consisting of Helichrysum arenarium flowers and the solvent using a regressive analysis for the theoretically predicted coordinates.
Materials and methods. For this research, milled officinal flowers of Helichrysum arenarium (Helichrysum arenarium L. flores) were used. The analysis of the extractions was carried out by RP HPLC method. Isosalipurposide, salipurposide, and chlorogenic acid of >98.0% purity were used as reference substances. The analytical wavelengths were 370, 290, and 325 nm. Results. The obtained experimental data are well-approximated by regressive linear equations in the theoretically predicted coordinates 1/C=f(V) and ln(b/a)=f(1/T). Wherein, the coefficient of determination of regressive equations was R2>0.998, which indicates functional dependence between the studied parameters and confirms the adequacy of the developed mathematical model. The experimental work identified the necessity of implementation of additional constant values into the mathematical model.
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 working hypothesis, mathematical models were developed and their adequacy was proved using a regressive analysis in the theoretically predicted coordinates. The results obtained could not deny that a mechanism of BAS distribution between the phases is explained and described by the classic Boltzmann distribution for discrete values of molecular energy (or quantum distribution according to Fermi and Dirac). Keywords: flowers of Helichrysum arenarium; isosalipurposide; salipurposide; chlorogenic acid; equilibrium; classic Boltzmann distribution for discrete values of molecular energy
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
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изучение распределения биологически активных
ВЕЩЕСТВ ИЗ ЦВЕТКОВ БЕССМЕРТНИКА ПЕСчАНОГО
между фазами экстракционной системы
Н.Н. Бойко1, Е.Т. Жилякова1, А.Ю. Малютина1, Д.К. Наплеков1, Н.Н. Шестопалова1, Д.С. Марцева1, О.О. Новиков2, Д.И. Писарев2, П.Г. Мизина3
1 Федеральное государственное автономное образовательное учреждение высшего образования «Белгородский государственный национальный исследовательский университет» Минобрнауки России 308015, Россия, г. Белгород, ул. Победы, 85
2 Центр коллективного пользования (научно-образовательный центр)
«Центр контроля качества лекарств», ФГАОУ ВО «Российский университет дружбы народов» 117198, Россия, г. Москва, ул. Миклухо-Маклая, 8/2
3 ФГБНУ «Всероссийский научно-исследовательский институт лекарственных и ароматических растений» 117216, Россия, г. Москва, ул. Грина, 7
E-mail: boykoniknik@gmail.com
Получено 16.09.2019 Рецензия (1) 10.10.2019 Рецензия (2) 15.10.2019 Принята к печати 21.10.2019
Цель. Проверка адекватности выдвигаемой рабочей гипотезы, которая объясняет и количественно описывает распределение БАВ в экстракционной системе из цветков бессмертника песчаного и растворителя, с помощью регрессионного анализа в предсказанных теорией координатах.
Материалы и методы. Для исследований использовали измельченное фармакопейное растительное сырье «Бессмертника песчаного цветки» (Helichrysum arenarium L. flores). Анализ извлечений проводили с помощью ОФ ВЭЖХ метода. В качестве стандартных веществ использовали изосалипурпозид, салипурпозид, хлорогеновую кислоту ФСО ГФУ, содержание >98,0 %. Аналитические длины волн 370, 290 и 325 нм.
Результаты. Экспериментальные данные хорошо аппроксимируются регрессионными линейными уравнениями в предсказанных теорией координатах 1/C=f(V) и ln(b/a)=f(1/T). При этом коэффициент детерминации регрессионных уравнений, имеет значение R2>0,998, что говорит о функциональной зависимости между изучаемыми параметрами и подтверждает адекватность разработанных уравнений. Эксперимент выявил необходимость введения в математическую модель дополнительной константы.
Заключение. Предложена рабочая гипотеза, которая объясняет и количественно описывает распределение БАВ в экстракционной системе из цветков бессмертника песчаного и этанола 80 % об. С помощью рабочей гипотезы разработаны математические модели, адекватность которых доказана с помощью регрессионного анализа в предсказанных теорией координатах. Полученные результаты не отвергают гипотезу, что механизм распределения БАВ между фазами в экстракционной системе объясняется и описывается классическим распределением Больцмана для дискретных значений энергии молекул (или квантовым распределением Ферми-Дирака).
Ключевые слова: цветки бессмертника песчаного; Helichrysum arenarium, изосалипурпозид; салипурпозид; хлоро-геновая кислота; равновесие; классическое распределение Больцмана для дискретных значений энергии молекул
INTRODUCTION
Flowers of Helichrysum arenarium (Helichrysum arenarium L. flores) are the officinal raw material within the territory of the Russian Federation, the Republic of Belarus, Ukraine, the Republic of Kazakhstan, etc. This raw material is used for the production of the drug product Flamin, which is manufactured in the form of tablets, granules and in-bulk substance. It is used in treatment of liver and gallbladder diseases. In addition, biologically active substances (BAS) from that raw material have antioxidant, antibacterial, antiviral, antihyperlipidemic, and cytotoxic effects [1-20].
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.
ФАРМАЦИЯ И ФАРМАКОЛОГИЯ
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.
Isosalipurposide, salipurposide, and chlorogenic acid of >98.0% purity were used as reference substances. Analytical wavelengths were 370, 290, and 325 nm.
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):
n
nn
1+ exp
AG kT
(1)
where n is quantity of BAS in the solvent with energy equals AG, mol;
n0 is an overall quantity of BAS, mol; AG 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.
1 1 „ — = — V + exp
AG kT
M
M
— =--V + KU--= a-V+b
n„
1Ц,
1Ц>
(2)
In Ku = In
AG
~k~
(3)
where C is concentration of BAS in the extracting solution, g/ml;
m0 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 (IVI/m0);
b is a constant that equals to multiplicity of Henry's constant and (KH) and the reversed value of overall BAS content in the raw material ^/m^, 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.
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Table 1 - The main validation parameters of analytical method and suitability of RP HPLC system
for the assay of isosalipurposide, salipurposide, and chlorogenic acid
Parameter Pharmacopoeia criteria [22] Isosalipurposide Salipurposide (sum of isomers) Chlorogenic acid
1. Retention time, min* - 20.1±0.2 11.9±0.2 and 12.8±0.2 6.3±0.3
2. Separation coefficient >1.5 3.0 3.1 and 3.2 11.0
3. Number of theoretical plates >1,000 103,458 39,541 and 29,267 12,282
4. Relative Standard <2.0 1.2 1.2 0.8
Deviation, RSD, %
5. LOD, g/mL - 4.1-10-5 5.540-6 2.2-10-5
6. LOQ, g/mL - 1.340-4 1.740-5 6.5-10-5
7. Determination coefficient, r2 >0.98 0.9999 0.9999 0.9999
8. Linear regressive
equation, C(g/ml) = - C=(2.79±0.06)-10-7\S C=(3.94±0.01)-10-7\S C=(2.92±0.04)40-7-S
/(S(mAU-sec))
* Note. The average value and its error (X±AX) were calculated on the basis of the repeat count n = 3 and the significance
level P = 0.95.
Table 2 - Constant values for BAS from Helichrysum arenarium flowers
Constant value
BAS AG, J/mol g m , % wt.
1. Isosalipurposide 5,390±380 -1.5±0.2 22.0±3.0
2. Salipurposide 19,930±1,030 -7.2±0.4 0.075±0.004
3. Chlorogenic acid 18,640±2,160 -5.4±0.9 0.45±0.08
Note. The number of experiments: n=4, the significance level: P=0.95.
Table 3 - Values of overall (initial) BAS content in the raw material (m0) found by the experiment and theoretical calculation
BAS Theoretically calculated content, m0/M, % wt.* Experimental values, m0/M, % wt.**
1. Isosalipurposide 1.58±0.06 1.46±0.07
2. Salipurposide 0.47±0.02 0.43±0.02
3. Chlorogenic acid 0.19±0.04 0.19±0.01
Note. * The number of experiments: n=4, the significance level: P=0.95. ** The number of experiments: n=3, the significance level: P=0.95.
DOI: 10.19163/2307-9266-2019-7-5-271-278
ФАРМАКОЛОГИЯ
3
Е
у
3000 2500 2000 1500 1000 500 0
Isosalipurposide
1/С = 63.7-V+ 99.9
R = 0.999 -60°С
1/С = 61.7V+109.9
R = 0.999 -40°С
1/С = 62.6-V+ 129.1
R = 0.999 -20°С
10
1/С = 65.3-V+ 151.5
R =0.999-4°С
20 30
V, ml
40
50
Figure 1 - Regressive equation of isosalipurposide concentration dependence on the volume of the extracting solution in coordinates 1/C=f(V)
w 1
У
12000 10000 8000 6000 4000 2000 0
1/C = 215.7-V + 223.8 R2 = 0.998- 60°C
Salipurposide
1/C = 204.8-V+329.6
R = 0.999 - 40°C
1/C = 212.0-V+577.6
R = 0.999 -20°C
10
1/C = 220.4-V + 978.2
R =0.998-4°C
20 30
V, ml
40
50
Figure 2 - Regressive equation of salipurposide concentration dependence on the volume of the extracting solution in coordinates 1/C=f(V)
00 1 и
35000 30000 25000 20000 15000 10000 5000 0
1/C = 461.8 V + 1723.5
Chbrogenic acid
498.9 V + 4713 999 - 20°С
R =0.998 - 4"C
10
20 30
V, ml
40
50
Figure 3 - Regressive equation of chlorogenic acid concentration dependence on the volume of the extracting solution in coordinates 1/C=f(V)
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PHARMACY& PHARMACOLOGY
3 2,5 2
it 1-5 _c
1 0,5
: 2242. lx-5.4285
R2 = 0.9986 Chlorogenic acid
y = 2396.6x - 7.17 R2 = 0.9997 Salipurposide
y = 647.81x-1.4931 R2 = 0.9995 Isosalipurposide
0,0028 0,003 0,0032 0,0034 0,0036 0,0038
1/T, 1/K
Figure 4 - Regressive equations of empiric constant values' (a, b) dependence on temperature in coordinates ln(b/a)=f(1/T) for BAS from Helichrysum flowers
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/C=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/100)) into the initial equation (1).
The constant values (AG, g and mp) 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 AG, which expresses the energy of the BAS interphase distribution,
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 (m0/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 (mjM), do not differ from each other. This fact additionally confirms the adequacy of the equation (2).
Hence, the obtained experimental results are well-complied 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:
1+ exp
AG
kT °J (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
ФАРМАЦИЯ И ФАРМАКОЛОГИЯ
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.
AUTHORS' CONTRIBUTION
All authors have equally contributed to the research work.
CONFLICT OF INTEREST
Authors state there's no conflict of interest between them.
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Nikolay N. Boyko - PhD (Pharmacy), Junior Researcher, Laboratory of Technology of Drugs, Associate Professor, Department of Pharmaceutical Technology, Belgorod National Research University, Russia, Belgorod. ORCID: 0000-0001-9222-2935. E-mail: boykoniknik@gmail.com
Elena T. Zhilyakova - Doctor of Sciences (Pharmacy), Professor, Head of the Department of Pharmaceutical Technology, Belgorod National Research University, Russia, Belgorod. E-mail: ezhilyakova@bsu.edu.ru.
Anastasiya Yu. Malyutina - PhD (Pharmacy), Associate Professor of the Department of Pharmaceutical Technology, Belgorod National Research University, Russia, Belgorod. ORCID: 0000-0001-6170-2151. E-mail: malyutina_a@bsu.edu.ru
Denis K. Naplekov - 2nd year postgraduate student, Belgorod National Research University, Russia, Belgorod. E-mail: 783767@bsu.edu.ru.
Natalia N. Shestopalova - PhD (Agricultural), Associate Professor of the Department of Pharmaceutical
Technology, Belgorod National Research University, Russia, Belgorod. E-mail: shestopalova@bsu.edu.ru.
Diana S. Martceva - 3rd year postgraduate student, Belgorod National Research University, Russia, Belgorod. E-mail: martseva@bsu.edu.ru.
Oleg O. Novikov - Doctor of Sciences (Pharmacy), Professor, Head of Test Department "Drugs quality control center" of the Shared Research and Education Center Peoples' Friendship University of Russia. ORCID: 0000-0003-3145-6783. E-mail: ole9222@yandex.ru
Dmitry I. Pisarev - Doctor of Sciences (Pharmacy), Professor of the Shared Research and Education Center, Peoples' Friendship University of Russia. ORCID: 00000002-2996-7712. E-mail: juniper05@mail.ru
Praskovia G. Mizina - Doctor of Sciences (Pharmacy), Professor, Vice-Director for Science of All-Russian Scientific Research Institute of Medicinal and Aromatic Plants. ORCID: 0000-0001-6510-9603. E-mail: mizina-pg@yandex.ru
