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ФАРМАЦИЯ И ФАРМАКОЛОГИЯ PHARMACY AND PHARMACOLOGY
®
УДК 54.03; 54.056; 547.458.88
J.M. landosov1, 2, 3<a. zh. Baimenov2,3, A. Sh. Iklasovai, Z.B. Sakipova1, N. Sakenova3, K. Laszlo4
1School of Pharmacy,AsfendiyarovKazakh National Medical University, 94Tole bi Street, Almaty, Kazakhstan 2lnstitute of Combustion Problems, 172 Bogenbay Batyr Street, Almaty, Kazakhstan 3Al-Farabi Kazakh National University, 71 Al-FarabiAvenue, Almaty, Kazakhstan 4Budapest University of Technology and Economics, Budapest, Hungary
Corresponding author E-mail: [email protected]
ISOLATION AND RHEOLOGICAL PROPERTIES OF PECTINS DERIVED FROM VEGETABLE
PULP
Resume: The purpose of this work is isolate and characterize pectins derived from (beet/apple and hawthorn pulpe), which are able to effectively remove heavy metals and other xenobiotics from the body in the course of enterosorption, upon oral administration. Each pectin type was isolated from a batch of 100 g of a corresponding starting raw material using acid extraction technique and the yields of each pectin, depending on the precursor, were compared with one another. Swelling testing, rheological properties and viscosity of the isolated pectins were studied and compared to each other. It was determined that the pectin obtained from sugar beet pulp/apple pomace exhibit low methylation/esterification ratio compared to that obtained from hawthorn pulp, which apparently has higher methyilation degree, and thus is more suitable for the purpose of higher uptake of heavy metal ions. Keywords: PECTIN, VEGETABLE PULP, ACID EXCTRACTION, SWELLING, RHEOLOGY, VISOSIMETRY, METHYILATION DEGREE
1. INTRODUCTION
Pectic polysaccharides are widely used in the medical industry due to their ability to form complexes with heavy metal ions and radionuclides [1].
On the other hand, sugar beet pulp produced at sugar plants in Kazakhstan do not undergo further processing into pectin and dietary fibers. To date, the market of Kazakhstan is not represented by the production of pectin, despite its widespread use in food technology, pharmaceutical production and medicine [2].Therefore, rational use freshly obtained sugar beet pulps, and pectin production is of the most immediate interest of Kazakhstan population with regard to design and boost local production of composite biopharmaceuticals with high added valueAcid hydrolysis is often used to extract pectins from plant materials. For hydrolysis-extraction of pectic polysaccharides, solutions of both mineral (nitric, hydrochloric, sulfuric) and organic (oxalic, lactic, citric) acids are used. Some authors used different conditions for the extraction of pectin, changing the pH of the medium, temperature and time: the pH of the medium varied from 1.0 to 2.5, the temperature of the reaction mixture - from 60 to 100 °C.It was found that with increasing extraction time, the temperature of the reaction medium should be reduced to 70-80 ° C. However, at temperatures above 50 °C and lowering the pH to 1.0, a significant hydrolysis of glycosidic bonds occurs, resulting in pectin with a low molecular weight and worse gelling capacity [3].
In experimental studies and clinical trials pectin's therapeutic efficiency in case of infectious pathology was
proved, which manifested as intoxication decrease, and also in case of intoxication with heavy metals, this is especially important for regions with high levels of pollution [4]. It is known that pectin substances have the ability to bind and remove from the human body stable and radioactive metals . Herewith, low-esterified pectin substances, which include sugar beet pectin, have the best complexing properties [1, 5, 6]. They also have the ability to increase the effectiveness of certain drugs, reduce their toxic effects on the body and eliminate some side effects. Used for medical purposes as additives to medicines, pectins must be of high purity[7].
2. EXPERIMENTAL
2.1. Methods for pectin isolation
100 g of sugar beet/apple pectinous pulpe (60/40 (wt/wt) ratio; "as received" from "Technologika" LTD (Ukraine) was transferred to Florence flask equipped with glass stopper and 2L of 1.5% of citric acid solution in distilled water (ca. pH -2.5) was added into the flask. The mixture was set into the thermostated bath and left for 48 h at 60 °C to digest. The extract solute was cooled and separated from the pulp through sedimentation and filtration in vacuum using Shott funnel and Bunsen flask.The filtrate (1.5 L) was concentrated by boiling off water down to 200 mL at ambient conditions. 300 ml of 96.6% ethanol was added to 200 ml of cooled extract in 1 L glass beaker for pectin to precipitate. The formed gelled suspension was transferred to 50 ml Eppendorf conical tubes and centrifuged for 15 minutes at 7000 rpm using Eppendorf® Centrifuge 5804, refrigirated at -4 °C. The eluent was decanted and pectin residue was
BECTHMK KA3HMY #4-2020-
washed 3 times with 70 % ethanol by resuspension/centrifugation. Upon drying at ambient conditions, the estmated yield of pectin is 5.5%. The same method was used to isolate howthorn pectin form dry howthorn: the yield is ca.2%.
2.2. Swelling test methodology
Initially, ca. 1 g of each pectin sample, dried at the ambient conditions, was thoroughly ground in agate mortar to ensure successive rapid water uptake during swelling/hydration test procedure. Each finely ground pectin sample powder in the amount of 1 ml was loaded onto a graduated glass measuring test tube, equipped with a stopper: both weighed/balanced. Then, 4 ml of water (the solvent) was added to each tube and left in the fridge for a couple of days to reach equilibrium of gelation, at 5°C in order to inhibit bacterial growth. The pectin gels could be visually observed to be syneretic and inhomogeneous, e.g.: both wetted pectin
samples in the test tubes formed translucent upper phases, which were pipetted out from opaque colored lower phases, the latter were then reweighted. The weight & volume measurements were undertaken to collect the corresponding data in order to be able to calculate both the equilibrium weight swelling ratio (ESR) and swelling volume ratio (Q).
2.3. Rheology and viscosity testing
Swollen pectin samples phases were measured with a plateplate geometry in rotation mode with a measuring gap of 0.5 mm, using Physica MCR 301 rheometer (Anton Paar GmbH, Austria).
3. RESULTS AND DISCUSSION Pectin isolation scheme for sugar beet/apple- and hawthorn pulpis shown in Figure 1.
Pectin Isolation Scheme
100 e of pectinous fruit/vegetable dry pulp
1 .Digestion with 2L of 1.5% citric acid aqueous solution (@60 °C for 48 h)
_Z_
2. Separation via sedimentation and
filtration to yield 1.5L of solute extract _±_
3. Solute extr act concentration via water
evaporation down from 1,5L to 200 mL _±_
4. Gelation precipitation of pectin using (3x100) mL of 96.6% ethanol per 200 ml of solute concentrate
_Z_
5. Centrifugation of precipitated pectin for 15 min at 7000 rpm @ -4°C followed by ale. eluent decantation
_±_
6. Washing of centrifueed pectin with 70% ethanol
(x 3 times) via resuspension/centrifugation _±_
7. Drying at ambient conditions to yield up to 5.5 % of pectins
Figure1 - Pectin isolation scheme for fruit/vegetable dry pulp
The swelling test measurements results for sugar beet/apple (SB/A)- and hawthorn (H)-pectins are summarized in Table 1. Table 1 - Swelling test measurements and results for pectins samples_
sample code mo pectin, g тн2о, g Mtotal, g Vup+L/ph, ml VL/ph, ml AVu/l ph, ml mswell, g ESR, % Q
SB/A-pectin 0.79 3.83 4.62 4.5 1.7 2.8 1.82 130 2.3
H -pectin 0.82 3.83 4.65 4.5 3.7 0.8 3.85 370 4.7
- m0 pectin - the absolute weight of a dried pectin sample, assessed by the difference between the weights of a measured graduated glass, sealed with a stopper (mglasstube + mm), with and without pectin sample;
- mH2o - the weight of added water at the equilibrium hydration point (mH2o = Mtotal- m0 pectin);
- Mtotal - the wetted pectin sample total weight (both upper and lower phases), e.g.: Mtotal=m0 pectin+mH2o;
- Vup+L/ph - the volume reading of a pectin sample total volume of both upper and lower phases;
- VL/ph, - the volume reading of a pectin sample lower phase, saturated with water;
- AVu/l ph - the volume of a pipetted out pectin sample upper phase, e.g.: AVu/l ph = Vup+L/ph - VL/ph;
- mswell - the lower phase weight of a swollen hydrated pectin sample at the equilibrium point.
ESR-value, which represents the ratio of a solvent weight to a polymer weight in a swollen polymer was calculated as follows:
ESR, % = [(mswell - m0 pectin)/m0 pectin ]*100%
and swelling volume ratio (Q) was calculated as follows:
Q = mswell/m0 pectin
When determining the viscosity by means of rheometers (viscometers) usually the shear rate is increased within a given period of time. The resulting shear stress is measured and displayed as flow curve. Viscosity curves of each pectin
are shown in the Figure 2. Viscosity valuesare calculatedfor each sample by linearization the function of Shear stress/Shear rate and areshown on flow curves (Figure 2 c, d).
130 120 110 100 90
I80
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30 20 10 о
1-'-'—......I
1 Shear Rate [1/s] m b
| SB/A-pectin-lower phase Average Viscosity=1.277 [Pa*s]
Ft=0.9984
V
»
I—
20
~I-'-1-'-1—'—Г
30 40 50 60 Shear Rate [1/s]
c
—r~
70
—Г-
80
~1—
90
a
d
Figure 2 - Viscosity and flow curves for SB/A and Hawthorn pectin samples:
(a)- Lg (Viscosity) vs Lg (Shear Rate); (b)-Lg(Shear Stress) vs Lg(Shear Rate); (c) - Shear Stress vs Shear Rate for SB/A-pectin lower phase sample; (d) - Shear Stress vs Shear Rate for Hawthorn -pectin lower phase sample.
The flow and viscosity curves may be different for diverse fluids. There are a few fluids such as water or oil showing a linear relation between shear stress and shear rate. Then, independent from shear stress, the viscosity is constant and just depending on temperature and pressure. Such fluids are referred to as Newtonian fluids.
As most food, pectin solutions do not follow this proportionality. If they are stressed with increasing shear rate a decrease of viscosity will be observed. With increasing shear stress these fluids thus become inviscid. This behaviour is referred to as non-Newtonian, structure-viscous (also shear-thinning or pseudoelastic). The reason is
CONCLUSION
Two different pectins were isolated from hawthorn pulp, sugar beet/apple pomace and characterize by rheology studies and swelling testing. Each pectin type was isolated from a batch of 100 g of a corresponding starting vegetable pulp using acid extraction technique and the yields of each pectin, depending on the precursor, were compared with one another.
Swelling testing, rheological properties and viscosity of the isolated pectins were studied and compared to each other. It
for example that the macromolecules or fibers orientate towards the shear movement in the measuring gap enabling the single particles to float past each other. From figure 2, it is evident , that SB/A-pectin sample express more Newtonian behavior compared to Hawthorn-pectin sample, which means that it is low-esterified having lesser methyilation degree compared to that of Hawthorn-pectin sample, and therefore is more suitable for heavy metal uptake in gastrointestinal tract. However Hawthorn-derived pectin can be further deesterifiedusing a simple technique via alkaline hydrolysis [1].
is evident that the rheological curves can be useful to model the behaviour of the swollen samples in the gastrointestinal tract as shear forces can occur there due to the peristaltics. The resulting pectins, upon modification have a potential and are designated to effectively remove heavy metals and other xenobiotics from the body in the course of enterosorption, upon oral administration.
1 Khotimchenko M. Lead-binding capacity of calcium pectates with different molecular weight / M. Khotimchenko, K. Makarova, E. Khozhaenko, V. Kovalev// International Journal of Biological Macromolecules. - 2017. - V. 97. - P. 526-535.
2 2.А.Ш. Икласова, З.Б. Сакипова, Э.Н. Бекболатова. Пектин: состав, технология получения, применение в пищевой и фармацевтической промышленности.// Вестник КазНМУ - 2018. - №3 - С. 243-244
3 Environmentally friendly preparation of pectins from agricultural byproducts and their structural./rheological characterization / B. Min, J. Lim, S. Ko et al. // Bioresource Technology. - 2011. - № 4. Vol. 102. - Pp. 3855-3860
4 Соболев М.Б. Особенности лечения токсического действия тяжелых металлов у детей.//Пути повышения
эффективности антидотной терапии. - СПб.: Институт токсикологии Минздрава России, 2004. - Т.5. - С. 191-198
5 Донченко Л.В., Фирсов Г.Г. Пектин: основные свойства, производство и применение. - М.: ДеЛипринт, 2007. -276 с.
6 IvnitskyYu.Yu., Krasnov K.A., Rejniuk V.L., Lapina N.V., Melikhova M.V., Golovko A.I., Krasnova A.A. Pectins in prevention of acute poisoning. // Toxicological Review. -2020 - Vol.2. - P. 30-35. (InRuss.) https://doi.org/10.36946/0869-7922-2020-2-29-34
7 Голыбин В.А., Матвиенко Н.А., Федорук В.А. Способ получения пищевых волокон из отхода свеклосахарного производства. // Инновационная наука. - 2015. - № 101. - С. 58-59.
*Ж.М. ЖАНДОСОВ1,2,з, А.Ж. БАЙМЕНОВ2,3, А.Ш. ИКЛАСОВА1, З.Б. САКИПОВА1, Н. САКЕНОВА3, К.
1Школа Фармации, НАО "Казахский Национальный Медицинский Университет" им. Асфендиярова, Алматы, Казахстан
2Институт Проблем Горения, Алматы, Казахстан 3Казахский Национальный Университет, Алматы, Казахстан 4Будапештский Университет Технологии и Экономики, Будапешт, Венгрия
ВЫДЕЛЕНИЕ И РЕОЛОГИЧЕСКИЕ СВОЙСТВА ПЕКТИНОВ, ПОЛУЧЕННЫХ ИЗ РАСТИТЕЛЬНОГО ЖОМА
Резюме: Целью данной работы является выделение и изучение свойств пектинов, полученных из растительной массы (свекловично-яблочный жом и жмых боярышника), которые способны эффективно удалять тяжелые металлы и другие ксенобиотики из организма в процессе энтеросорбции при пероральном приеме. Каждый тип пектина выделяли из партии в 100 г соответствующего исходного сырья с использованием метода кислотной экстракции, и выходы каждого
пектина, в зависимости от предшественника, сравнивали друг с другом. Были поведены тесты на набухание, изучены реологические свойства и вязкость выделенных пектинов и произведено сравнение образцов. Было определено, что пектин, полученный из жома сахарной свеклы/ яблочного жома, демонстрирует низкое соотношение метилирования/этерификации по сравнению с пектином, полученным из жома боярышника, который, по-видимому, имеет более
высокую степень метилирования и, таким образом, больше подходит для цели эффективного связывания ионов тяжелых металлов.
Ключевые слова: ПЕКТИН, РАСТИТЕЛЬНАЯ МАССА, КИСЛОТНАЯ ЭКСТРАКЦИЯ, РЕОЛОГИЯ, ВИСКОЗИМЕТРИЯ, СТЕПЕНЬ МЕТИЛИРОВАНИЯ
*Ж.М. ЖАНДОСОВ1,2 3, А.Ж. БАЙМЕНОВ2,3, А.Ш. ИКЛАСОВА1, З.Б. САКИПОВА1, Н. САКЕНОВА3, К.
1Асфендияров атындагы Цазац улттыцмедициналыцуниверситету Фармация мектебу Алматы, Цазацстан 2Жану проблемалары институты, Алматы, Цазацстан 3эл-Фараби атындагы Цазац улттыцуниверситету Алматы, Цазацстан 4Будапешт технология жэне экономика университету Будапешт, Венгрия
0СШД1КТЕН АЛЬЮТАН ПЕКТИНДЕРД1 ОКДОАУ ЖЭНЕ РЕОЛОГИЯЛЬЩ КДСИЕТТЕР1Н ЗЕРТТЕУ
Туши: Бул жумыстыщмацсаты - энтеросорбция арцылы ауыр металдарды жэне басца ксенобиотиктердi организмнен тиiмдi турде шыгаруга цабiлеттi есмдЫ заттарынан (цызылша/алма жэне долана целлюлозасы) алынган пектиндердi белт алу жэне сипаттау. Пектиннщ эр тyрi цышцылды экстракциялау эдс арцылы 100 г сэйкес шиюзат партиясынан белтт алынды жэне эр пектиннщ енШ, iзашарына байланысты, бiр-бiрiмен салыстырылды. Оцшауланган пектиндердщ iсiнуi, реологиялыц цасиеттерi жэне тутцырлыгы сынацтары зерттелт, бiр-бiрiмен
салыстырылды. К,ант цызылшасы целлюлозасынан/ алма тортынан алынган пектин метилдену дэрежеа жогары болып кертетт жэне сондыцтанауырметалды иондар жогары сщру мацсаттарына цолайлы долана шогырынан алынган пектинмен салыстырганда темен метилдену/эфирлену коэффициенты керсететiнi аныцталды.
Туй^ свздер: ПЕКТИН, 6С1МШ1Л1К МАССА, ЦЫШКЫЫЛДЫ СЫРЫП АЛУ, РЕОЛОГИЯ, ВИЗКОСИМЕТРИЯ, МЕТИЛДЕУ ДЭРЕЖЕС1
