Визначення фітохімічного складу субстанції Альтабор
The development of medicines based on alder cone extracts led to the introduction of Altan and Altabormedicines into medical practice. The technology of extraction cake from cones has made it possible to obtain extracts with different therapeutic properties.Aim. To develop an effective method for stud...
Збережено в:
| Опубліковано в: | Журнал органічної та фармацевтичної хімії |
|---|---|
| Дата: | 2021 |
| Том: | 19 |
| Випуск: | 1(73) |
| Сторінки: | 16-24 |
| ISSN: | 2518-1548 |
| Автори та афіліації: |
|
| Автори: | , |
| Формат: | Стаття |
| Мова: | Англійська |
| Опубліковано: |
National University of Pharmacy
2021
|
| Теми: | |
| Онлайн доступ: | https://ophcj.nuph.edu.ua/article/view/202081 |
| Теги: |
Додати тег
Немає тегів, Будьте першим, хто поставить тег для цього запису!
|
| Назва журналу: | Journal of Organic and Pharmaceutical Chemistry |
| Завантажити файл: |
|
Репозитарії
Journal of Organic and Pharmaceutical Chemistry| _version_ | 1874455125713485824 |
|---|---|
| author | Sheiko, S. Yu. Shalamay, A. S. |
| author_facet | Sheiko, S. Yu. Shalamay, A. S. |
| author_institution_txt_mv | [
{
"author": "S. Yu. Sheiko",
"institution": "V. P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of Ukraine",
"orcid": ""
},
{
"author": "A. S. Shalamay",
"institution": "PJSC SIC “Borshchahivskiy CPP”",
"orcid": ""
}
] |
| author_sort | Sheiko, S. Yu. |
| baseUrl_str | https://ophcj.nuph.edu.ua/oai |
| collection | OJS |
| container_end_page | 24 |
| container_issue | 1(73) |
| container_start_page | 16 |
| container_title | Журнал органічної та фармацевтичної хімії |
| container_volume | 19 |
| datestamp_date | 2026-08-24T15:33:48Z |
| description | The development of medicines based on alder cone extracts led to the introduction of Altan and Altabormedicines into medical practice. The technology of extraction cake from cones has made it possible to obtain extracts with different therapeutic properties.Aim. To develop an effective method for studying the qualitative composition of the Altabor substance anddetermine the quantitative content of its components.Results and discussion. The Altabor substance is a complex mixture of ellagitannins containing more than 70 components. The main components of the extract (or their isomers) were determined by mass spectrometry and by comparing the retention times with the literature data. Gallic, ellagic, valoneic acids dilactone were conclusively determined by adding reference standards of these acids to the extract. The substance contains the following compounds: 2,3-hexahydroxydiphenoyl-(α/β)-glucose tr1 = 0.55 min, tr2 = 0.89 min (α and β isomers), 4,6-O-[(S)-valeonyl]-D-glucose (isomer) tr = 0.64 min, gallic acid tr = 1.198 min, pedunculagin tr1 = 3.63 min, tr2 = 4.62 min(α and β isomers), proecoxin A (isomer) tr = 4.78 min, valoneic acid dilactone tr = 6.19 min, ellagic acid pentoside (isomer) tr = 7.07 min, ellagic acid tr = 7.335 min.Experimental part. The composition analysis was performed using an Agilent 1200 chromatograph with a UV detector, a G6140 mass detector, an Alltech 3300 light scattering detector (ELSD), as well as the Agilent ChemStation Rev.B.04.03 software. The molecular weights of the compounds in the extract were determined using the method of mass spectrometry of ESI-electrospray ionization. The determination of the components was performed using an ultraviolet detector at a wavelength of 280 nm. The column was Rapid Resolution HT Cartige, 4.6 × 30 mm, 1.8 μm, Zorbax SB-C18.Conclusions. A new effective method of analysis of the Altabor substance has been developed; it allowsdetermining the qualitative and quantitative content of its structural components. The method gives the possibility to control the process of obtaining the Altabor substance, study the dependence of its composition on the conditions of its obtaining, batch number, place, time of the natural raw material collection, and study the composition of other pharmaceutical substances, the plant raw material containing tannins. The advantage of the method isthe short time (up to 10 min) of analysis using high-performance liquid chromatography at high resolution.Key words: gallotannins; ellagitannins; Altabor; chemical composition; structure; HPLC (high-performance liquid chromatography) |
| doi_str_mv | 10.24959/ophcj.21.202081 |
| first_indexed | 2025-07-17T13:00:47Z |
| format | Article |
| fulltext |
Journal of Organic and Pharmaceutical Chemistry. – 2021. – Vol. 19, Iss. 1 (73)
16
ISSN 2308-8303 (Print) ISSN 2518-1548 (Online)
UDC 615.322.074:581.192:582.622.1 https://doi.org/10.24959/ophcj.21.202081
S. Yu. Sheiko1, A. S. Shalamay2
1 V. P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the National Academy
of Sciences of Ukraine, Ukraine
1, Murman’ska str., Kyiv, 02660, Ukraine. E-mail: ssheiko@ukr.net
2 PJSC SIC “Borshchahivskiy CPP”, Kyiv, Ukraine
The determination of the phytochemical composition
of the Altabor substance
The development of medicines based on alder cone extracts led to the introduction of Altan and Altabor
medicines into medical practice. The technology of extraction cake from cones has made it possible to obtain
extracts with different therapeutic properties.
Aim. To develop an effective method for studying the qualitative composition of the Altabor substance and
determine the quantitative content of its components.
Results and discussion. The Altabor substance is a complex mixture of ellagitannins containing more than
70 components. The main components of the extract (or their isomers) were determined by mass spectrometry
and by comparing the retention times with the literature data. Gallic, ellagic, valoneic acids dilactone were conclu-
sively determined by adding reference standards of these acids to the extract. The substance contains the following
compounds: 2,3-hexahydroxydiphenoyl-(α/β)-glucose tr1 = 0.55 min, tr2 = 0.89 min (α and β isomers), 4,6-O-[(S)-
valeonyl]-D-glucose (isomer) tr = 0.64 min, gallic acid tr = 1.198 min, pedunculagin tr1 = 3.63 min, tr2 = 4.62 min
(α and β isomers), proecoxin A (isomer) tr = 4.78 min, valoneic acid dilactone tr = 6,19 min, ellagic acid pentoside
(isomer) tr = 7.07 min, ellagic acid tr = 7.335 min.
Experimental part. The composition analysis was performed using an Agilent 1200 chromatograph with
a UV detector, a G6140 mass detector, an Alltech 3300 light scattering detector (ELSD), as well as the Agilent
ChemStation Rev.B.04.03 software. The molecular weights of the compounds in the extract were determined
using the method of mass spectrometry of ESI-electrospray ionization. The determination of the components
was performed using an ultraviolet detector at a wavelength of 280 nm. The column was Rapid Resolution
HT Cartige, 4.6 × 30 mm, 1.8 μm, Zorbax SB-C18.
Conclusions. A new effective method of analysis of the Altabor substance has been developed; it allows
determining the qualitative and quantitative content of its structural components. The method gives the possibility
to control the process of obtaining the Altabor substance, study the dependence of its composition on the condi-
tions of its obtaining, batch number, place, time of the natural raw material collection, and study the composition
of other pharmaceutical substances, the plant raw material containing tannins. The advantage of the method is
the short time (up to 10 min) of analysis using high-performance liquid chromatography at high resolution.
Key words: gallotannins; ellagitannins; Altabor; chemical composition; structure; HPLC (high-performance
liquid chromatography)
С. Ю. Шейко1, А. С. Шаламай2
1 Інститут біоорганічної хімії та нафтохімії імені В. П. Кухаря НАН України, Україна
2 ПАТ НВЦ «Борщагівський ХФЗ», Україна
Визначення фітохімічного складу субстанції Альтабор
Розробка лікарських засобів на основі екстрактів суплідь вільхи вперше завершилась впровадженням
у медичну практику препаратів Альтан та Альтабор. Особливості технології екстрагування підготовленого
шроту суплідь дозволило отримувати екстракти з різними терапевтичними властивостями.
Мета. Розробити ефективний метод вивчення якісного складу субстанції Альтабор із визначенням
кількісного вмісту компонентів.
Результати та їх обговорення. Субстанція Альтабор становить собою складну багатокомпонентну суміш
елаготанінів, що містить більше 70 компонентів. Основні компоненти екстракту визначено за допомогою мас-
спектрометрії, а також шляхом порівняння часів утримування з літературними даними. Галову, елагову,
валонову кислоти визначено остаточно шляхом додавання в екстракт стандартів цих кислот. До його складу
входять ідентифіковані сполуки: 2,3-гексагідроксидифеноїл-(α/β)-глюкоза tr1 = 0,55 хв, tr2 = 0,89 хв (α та
β ізомери), 4,6-O-[(S)-валеоніл]-D-глюкоза (ізомер) tr = 0,64 хв, галова кислота tr = 1,198 хв, педункулагін
tr1 = 3,63 хв, tr2 = 4,62 хв (α та β ізомери), праекоксін А (ізомер) tr = 4,78 хв, дилактон валонової кислоти
tr = 6,19 хв, пентозид елагової кислоти (ізомер) tr = 7,07 хв, елагова кислота tr = 7,335 хв.
Експериментальна частина. Аналіз складу проводили за допомогою Agilent 1200 хроматографа з УФ-
детектором, мас-детектором G6140 та детектором світлорозсіювання (ELSD) Alltech 3300. Програмне забезпе-
чення Agilent ChemStation Rev.B.04.03. Молекулярні маси сполук, які входять до складу екстракту, визначе-
но за допомогою методу мас-спектрометрії іонізацією розпиленням в електричному полі (ESI-electrospray
ionization). Визначення компонентів проводили із застосуванням ультрафіолетового детектора за довжи-
ни хвилі λ 280 нм. Колонка: Rapid Resolution HT Cartige 4,6 × 30 мм, 1,8 мкм, Zorbax SB-C18.
Висновки. Розроблено новий ефективний метод аналізу фармсубстанції Альтабор, який дозволяє
вивчати її якісний склад та визначати кількісний вміст компонентів. Метод дозволяє контролювати процес
виробництва субстанції Альтабор, вивчати залежність її складу від умов отримання, номеру партії, місця,
часу збору природної сировини, вивчати склад інших фармсубстанцій, природної сировини, що містить та-
ніни. Перевага методу – короткий час (до 10 хвилин) проведення аналізу за допомогою високоефективної
рідинної хроматографії за високої роздільної здатності.
Ключові слова: галотаніни; елаготаніни; Альтабор; хімічний склад; структура;
ВЕРХ (високоефективна рідинна хроматографія)
Copyright © 2021, S. Yu. Sheiko, A. S. Shalamay
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0)
Журнал органічної та фармацевтичної хімії. – 2021. – Т. 19, вип. 1 (73)
17
ISSN 2518-1548 (Online) ISSN 2308-8303 (Print)
European alder (Alnus glutinosa) and grey alder
(Alnus incana) are found almost on the whole terri-
tory of Ukraine, especially on the forest lowland wet-
lands and floodplains of many small and large rivers
and firstly in the Polissia area [1].
Since ancient times, the interest of folk medicine
to this plant has been associated with the healing pro-
perties of its bark and cones. The high content of tan-
nins, namely gallo- and ellagitannins, reveals the thera-
peutic effect of tinctures and decoctions from the parts
of this plant [2].
The development of medicines based on alder cone
extracts has led to the introduction of such medici-
nes as Altan and Altabor into medical practice. The tech-
nology of extraction cake from cones has made it pos-
sible to obtain extracts with different therapeutic pro-
perties [3, 4].
Thus, the use of 75 % aqueous ethanol under the con-
ditions of filtration extraction of European alder (Alnus
glutinosa) infructescence and grey alder (Alnus incana)
allows obtaining a dry extract for the preparation of
Altan medicine prescribed for diseases of the diges-
tive tract of different localization – for the complex
treatment of gastric ulcers and various sections of
the stomach and small and large intestine, as well
as infections of the digestive tract [5 – 9]. The hepato-
protective effect of Altan medicine in the case of acute
and chronic liver lesions is quite pronounced and ma-
nifests itself even when using the drug in low doses [10].
The drug tablet contains 10 mg of the Altan extract cal-
culated with reference to dried substance, and the
content of ellagitannins amounts to 60 % in it.
Altabor medicine is produced on the basis of
the aqueous extract of European alder (Alnus glutinosa).
The active ingredients of the dry extract – gallo- and
ellagitannins in the composition of the drug have
a marked antiviral effect against influenza viruses and
herpes. This drug, which tablet contains 20 mg of
the dry extract, is used in the treatment of influenza,
acute respiratory viral infections, and for the preven-
tion of these diseases [3, 4, 11].
The active agents of both substances of the dry
extract of the European alder infructescence exhibit
a pronounced antioxidant, anti-inflammatory and mem-
brane-stabilizing action, which complements their
pharmacological effects [3, 4].
The technological processes of obtaining these dry
extracts in the production of both drugs may alter
their true phytochemical composition, and this may
be due to their ability to easily hydrolyze gallo- and
ellagitannins, constituents of alder cones (Fructus Alni).
In connection with this, we should further study
the natural phytochemical composition of biologically
active substances of European alder, in particular tan-
nins, which, in fact, has not been studied with some
exceptions [12]. It is also important to examine the sub-
stance from the alder raw material – Altabor since its
constant composition of active ingredients, namely
tannins, will enable to achieve a guaranteed perma-
nent pharmacological effect of drugs from this sub-
stance. The paper [12] presents the isolation of three
substances – tannins in the extract from cones by four
preparative HPLC methods. The substances were ob-
tained in very small amounts of 1 – 8 mg. The quali-
tative HPLC chromatograms of alder cone extracts,
which would determine their component composi-
tion, are not described in the literature. Moreover, in
Ukraine, the studies to determine the phytochemical
composition of the natural raw material containing
hydrolyzable tannins, as well as pharmaceuticals ma-
nufactured based on this raw material, have not been
carried out at all. Only the total tannin content was
determined. Altabor and Altan have been manufac-
tured since 1990s by the PJSC SIC “Borshchahivskiy
CPP”. The company previously made attempts to ob-
tain high-quality HPLC chromatograms, which would
allow studying the qualitative composition of these
substances, as well as the raw material, and deter-
mine the quantitative content of components, but all
of them were unsuccessful [13]. The phytochemical
composition of the Altabor substance is unknown.
To date, the quality of the Altabor substance is deter-
mined by measuring the total amount of tannins by
spectrophotometry. The raw material, i.e. the alder co-
nes, has been studied similarly. It is important to find
the phytochemical composition of the raw material
since its composition can be influenced by such fac-
tors as soil properties, the time of the raw material
collection, climatic and other conditions under which
alder grows [14]. This can affect the manufacturing
process. To separate a mixture containing more than
70 substances with the similar chromatographic pro-
perties is a complicated task [15]. Therefore, the re-
sults presented in this paper are important.
Thus, the aim of this work was to develop an ef-
fective method for studying the qualitative composi-
tion of the Altabor substance and determine the quan-
titative content of its components.
To determine the composition, the crushed sam-
ple of the Altabor substance (batch No. 301217) was
extracted with methyl alcohol. As a method of analy-
sis of the extract obtained the method of high-perfor-
mance liquid chromatography using an Agilent 1200
chromatograph was proposed.
The extract is a complex multicomponent mixture
of tannins with very similar chromatographic proper-
ties, which is difficult to separate. In order to select
the conditions for successful separation of the extrac-
ted ellagitannins, a suitable chromatographic column
and solvent systems for elution, as shown in the ex-
Journal of Organic and Pharmaceutical Chemistry. – 2021. – Vol. 19, Iss. 1 (73)
18
ISSN 2308-8303 (Print) ISSN 2518-1548 (Online)
perimental part, were selected. On the way to achieve
the desired result, namely the creation of the HPLC
method that would allow determining the Altabor sub-
stance composition, a number of isocratic solvent
systems were tested, for example: H2O:CH3CN in the ra-
tio of 90:10; H2O:CH3CN:THF:HCOOH in the ratio of
70:5:25:0.2; H2O:CH3CN:THF:HCOOH in the ratio of
90:5:5:0.2; 0.1N H3PO4:0.1N KH2PO4:CH3CN in the ra-
tio of 42.5:42.5:15; H2O:CH3CN:THF:HCOOH in the ra-
tio of 80:5:15:0.2, as well as gradient systems, for
example: H2O:CH3CN with various linear gradients –
0 – 30; 0 – 50; 0 – 100 and others. However, the systems
presented in the article turned out to be much better.
For the analysis of such polar substances as tan-
nins it is better to use the chromatographic columns
filled with C18 modified silica gel. One should choose
the smallest size of particles among available – 1.8 μm.
Resolution decreases when 5 μm columns are used.
According to the results of the chromatographic
and mass spectrometric studies the Altabor substan-
ce is a complex mixture of ellagitannins containing more
than 70 components (see chromatogram, Fig. 1). The main
components of the substance (or their isomers) were
determined by mass spectrometry and by comparing
the retention times with the literature data [16 – 18].
Gallic (3), ellagic (8), valoneic acid dilactone (6) were
conclusively determined by adding reference stan-
dards to the substance. The reference standard of valo-
neic acid dilactone with a purity of more than 98 % was
obtained by the method described in the experimen-
tal part. Its 1H NMR spectrum and HPLC are shown in
Fig. 14, 15. To determine gallic and ellagic acids, com-
mercially available Sigma-Aldrich samples were used.
The HPLC-MS experiments revealed that the Alta-
bor substance contains the following compounds
(Fig. 1, 2): 2,3-hexahydroxydiphenoyl-(α/β)-gluco-
se (1), tr1 = 0.55 min, tr2 = 0.89 min (α and β isomers)
(Fig. 3); 4,6-O-[(S)-valeonyl]-D-glucose (an isomer) (2),
tr = 0.64 min (Fig. 4); gallic acid (3), tr = 1.198 min;
pedunculagin (4), tr1 = 3.63 min, tr2 = 4.62 min (α and
β isomers) (Fig. 5)а; praecoxin A (5) (an isomer),
tr = 4.78 min (Fig. 6); valoneic acid dilactone (6),
а Mass spectra of the compounds with tr =3.63 min and 4.62 min are the same. The areas of two peaks with tr = 3.63 min and tr = 4.62 min
are almost identical. Having compared the retention times with the literature data we can conclude that two peaks on the chromatogram
probably belong to the same substance – α and β pedunculagine isomers (4).
Fig. 1. The chromatogram of the Altabor substance (Batch No. 301217). Methanol Extract. System I. (A) 0.1 % HCOOH (aqueous solution),
(B) CH3CN. 0-1.5 min, 100 % A (isocratic mode); 1.5 – 7.5 min, 0-20 % B in A (linear gradient); flow-rate – 1 mL/min, 280 nm
Журнал органічної та фармацевтичної хімії. – 2021. – Т. 19, вип. 1 (73)
19
ISSN 2518-1548 (Online) ISSN 2308-8303 (Print)
OH
OH
OHHOOHHO
HO
HO
HO
O
O
O
2,3-HHDP-glucose ( )1
HO
HO
HO
OH
HO
OH
OH
HO
O
O
O
O
HO
HO
OH
CO2H
4,6-Valoneoylglucose ( )2
CO2HHO
HO
HO
Gallic acid ( )3
HOOHHO
HO
OH
OH
HO
HO
HO
OH
HO
OH
O
O
OH
O
O
O
Pedunculagin ( )4
HOOHHO
HO
OH
OH
HO
HO
HO
OH
HO
OH
O
O
O
O
O
OH
OH
OH
HO2C
O
Praecoxin A ( )5
O
O
O
O
OH
HO
HO O
HO OH
OH
HO2C
Valoneic acid dilactone ( )6
O
O
O
O
OH
HO
HO OR
R = xylose
Ellagic acid 4-O-xyloside ( )7Ellagic acid ( )8
O
O
O
O
OH
HO
HO OH
O
O
O
O
O
O
O
O
O
O
O
O
Fig. 2. The main components of the Altabor substance
Fig. 3. The negative ion mass spectrum of the compound 1
Fig. 4. The negative ion mass spectrum of the compound 2
Fig. 5. The negative ion mass spectrum of the compound 4
Fig. 6. The negative ion mass spectrum of the compound 5
Fig. 7. The negative ion mass spectrum of the compound 7
Journal of Organic and Pharmaceutical Chemistry. – 2021. – Vol. 19, Iss. 1 (73)
20
ISSN 2308-8303 (Print) ISSN 2518-1548 (Online)
tr = 6.19 min; ellagic acid pentoside (most probably
xyloside, an isomer) (7), tr = 7.07 min (Fig. 7); ella-
gic acid (8), tr = 7.335 min. The Altabor extract also
contains a mixture of hexoses and possibly pentoses,
which are not fixed by the UV detector and are not re-
tained by the C-18 column phase. These compounds
can be detected on a light scattering detector at the be-
ginning of the chromatograms with tr = 0.3 min in
the form of a mixture.
The Altabor substance was additionally fraction-
ated by column chromatography with a Diaion HP-20
sorbent. While fractioning the starting substance gave
fractions containing much less number of compounds
than the raw material. Thus, it is easier to analyze such
mixtures. It becomes possible to obtain the minor com-
pounds characteristics, which small peaks are hidden
by other peaks on the raw material chromatogram.
Moreover, re-determination of the molecular masses
in fractions is an additional proof of the presence of
the compounds previously found.
As an eluent the aqueous solution of ethyl alcohol
of different concentrations was used. The concentra-
tion of ethyl alcohol was increased stepwise. As the re-
sult 5 fractions were obtained. The fraction analysis
was performed by HPLC, in particular fraction 1 was
obtained by elution of the Altabor substance with water
Fig. 8. The chromatogram of the Altabor substance fraction 1
(Diaion HP-20, pure water) (system I)
Fig. 9. The chromatogram of the Altabor substance fraction 2
(Diaion HP-20, 10 % EtOH) (system I)
Fig. 10. The chromatogram of the Altabor substance fraction 3
(Diaion HP-20, 20 % EtOH) (system I)
Fig. 11. The chromatogram of the Altabor substance fraction 4
(Diaion HP-20, 40 % EtOH) (system I)
Журнал органічної та фармацевтичної хімії. – 2021. – Т. 19, вип. 1 (73)
21
ISSN 2518-1548 (Online) ISSN 2308-8303 (Print)
(Fig. 8), fractions 2, 3, 4, 5 by elution with 10 %, 20 %,
40 % and 96 % ethyl alcohol, respectively (Fig. 9 – 12).
The order of the substance release (ellagitannins
from the column filled with Diaion HP-20) was simi-
lar to the order of the substance release from the HPLC
column filled with a sorbent – C18 modified silica gel.
The main components of the fraction 1 (Fig. 8) were 2,3-
hexahydroxydiphenoyl-(α/β)-glucose (1), tr1 = 0.63 min,
tr2 = 1.08 min (α and β isomers), 4,6-O-[(S)-valeonyl]-
D-glucose (2) (an isomer), tr = 0.76 min, gallic acid
(3), tr = 1.46 min; the fractions 2 and 3 (Fig. 9 and
Fig. 10, respectively) – pedunculagin (4), tr1 = 3.84 min,
tr2 = 4.81 min (α and β isomers), praecoxin A (5),
tr = 4.96 min; fractions 4 and 5 – ellagic acid (8) and
Fig. 12. The chromatogram of the Altabor substance fraction 5
(Diaion HP-20, 96 % EtOH) (system I)
Fig. 13. HPLC repeated chromatographic (Diaion HP-20, 20 %
EtOH) (system I) separation of fraction 3 (elution with 20 % EtOH)
Fig. 14. 1Н NMR spectrum of valoneic acid dilactone in DMSO-d6
Journal of Organic and Pharmaceutical Chemistry. – 2021. – Vol. 19, Iss. 1 (73)
22
ISSN 2308-8303 (Print) ISSN 2518-1548 (Online)
valoneic acid dilactone (9), ellagitannins with mo-
lecular weights of 934 Da and 936 Da.
The main components of the purified fraction 3
of tannins were pedunculagin (4) and praecoxin A
(an isomer) (5) (Fig. 13). The resulting concentrate is
promising for further isolation of these compounds
required as standards for the study of their quanti-
tative content in the Altabor substance.
A preparative method for valoneic acid dilactone
isolation from the Altabor substance hydrolyzed in
acidic conditions was also developed. It can be used
to obtain a reference standard of this compound.
The 1H NMR spectrum of the valoneic acid dilactone
isolated and its HPLC are shown in Fig. 14 and 15,
respectively.
Experimental part
Equipment
The composition analysis was performed using
an Agilent 1200 chromatograph with a UV detector,
a G6140 mass detector, an Alltech 3300 light scattering
detector (ELSD), as well as the Agilent ChemStation
Rev.B.04.03 software.
The molecular weights of the compounds in the extract
were determined using the method of mass spectro-
metry of ESI-electrospray ionization – a gentle ioni-
zation method that allowed obtaining molecular ion
peaks of complex ellagitannin molecules without de-
stroying them. Formic or trifluoroacetic acid should
be used to ionize the molecules in the extract, as their
use improves the resolution. One can also use ammo-
nium acetate or ammonium formate.
The determination of the components was per-
formed using an ultraviolet detector at a wavelength
of 280 nm.
The column was Rapid Resolution HT Cartige,
4.6 × 30 mm, 1.8 μm, Zorbax SB-C18.
1H NMR spectrum was taken with a Varian VNMRS
400 MHz spectrometer, using TMS as an internal stan-
dard in DMSO-d6 solution.
Mobile phase systems
System I: (A) 0.1 % HCOOH (aqueous solution), (B)
CH3CN. 0 – 1.5 min, 100 % A (isocratic mode); 1.5 – 7.5 min,
0 – 20 % B in A (linear gradient); flow-rate – 1 mL/min.
System II: (A) 0.05 % CF3COOH (aqueous solu-
tion), (B) CH3CN. 0 – 1.5 min, 100 % A (isocratic mode);
1.5 – 7.5 min 0-20 % B in A (linear gradient); flow-
rate – 1 mL/min.
System III: (A) 0.1 % HCOOH, (B) CH3CN. 0 – 1.5 min,
100 % A (isocratic) 1.5 – 3 min, 0-5 % B in A (linear
Fig. 15. HPLC of the valoneic acid dilactone reference standard
Журнал органічної та фармацевтичної хімії. – 2021. – Т. 19, вип. 1 (73)
23
ISSN 2518-1548 (Online) ISSN 2308-8303 (Print)
gradient); 3 – 4.5 min, 5 % B (isocratic); 4.5 – 9 min,
5 – 20 % B in A (linear gradient); flow-rate – 1 mL/min.
System IV: (A) 0.1 % HCOOH (aqueous solution),
(B) CH3CN. 0 – 3 min, 100 % A (isocratic); 1.5 – 7.5 min,
0-20 % B in A (linear gradient); flow-rate – 1 mL/min.
Preparation of the methanol extract of the Alta-
bor substance. The sample of of the Altabor substance
(6.0 g) was mixed with 120 mL of methyl alcohol for
3 h. The dark solution was filtered, and the solvent
was evaporated in vacuum at a temperature below
40 °C. A dark solid product was obtained. The yield
was 4.2 g.
Fractionation of the Altabor substance with
Diaion HP-20 sorbent. A column: inner diameter
d = 1.5 cm, total volume v = 70 mL, the height of the sor-
bent column h = 25.5 cm, weight of the resin – 20 g.
The Altabor substance (700 mg) was dissolved in 1.5 mL
of distilled water with stirring. The solution was fil-
tered and applied to the top layer of the sorbent. Elution
of the column was carried out at a rate of 1.5 mL per
minute. The first elution was carried out with pure
water. Fraction 1 was collected in a volume of 110 – 120 mL.
Then elution by 10 %, 20 %, 40 %, 96 % ethyl alcohol
was successively carried out. Thus, fractions 2, 3, 4, 5
in volumes of 110 – 120 mL were collected. All frac-
tions were evaporated in vacuum at 20 mmHg on
a water bath with a temperature below 40oC.
Valoneic acid dilactone (reference standard).
The Altabor substance (2 g) was dissolved in the
ethyl alcohol-water mixture (1:2). Then 7 mL of the
concentrated hydrochloric acid were added to the so-
lution, and it was refluxed for 4 h. After the resin was
separated, the solvents were evaporated. As the re-
sult, a crystalline product, being preferably a mixture
of valoneic acid dilactone and ellagic acid, was ob-
tained. The resulting product (900 mg) was refluxed
for 30 min in 10 mL of dioxane. The hot solution
was filtered, while valoneic acid dilactone remained
in the solution. The filtrate was left to crystallize.
The precipitate was filtered. The procedure was re-
peated 3 – 4 times to obtain valoneic acid dilactone
with a purity of more than 98 % (HPLC control). The
yield was 50 mg.
Conclusions
A new effective method of analysis of the Altabor
substance has been developed; it allows determining
the qualitative and quantitative content of its struc-
tural components. The method gives the possibility
to control the process of obtaining the Altabor sub-
stance, study the dependence of its composition on
the conditions of its obtaining, batch number, place,
time of the natural raw material collection, and study
the composition of other pharmaceutical substances,
the plant raw material containing hydrolysable tan-
nins. The advantage of the method is the short time
(up to 10 min) of analysis using high-performance
liquid chromatography at high resolution.
Conflict of interests: the authors have no con-
flict of interests to declare.
References
1. Екофлора України; Дідух, Я. П., Ред.; Фітосоціоцентр: Київ, 2004; Т. 2, 442 – 447.
2. Задорожный, А. М.; Кошкин, А. Г.; Соколов, С. Я.; Шретер, А. И. Справочник по лекарственным растениям; Лесная промышленность:
Москва, 1981.
3. Рибалко, С. Л. Вивчення антивірусної активности препарату Альтабор на експериментальній моделі інфекції, спричиненої вірусом
гепатиту С, в культурі клітин. Вісник фармакології та фармації 2010, 3, 29 – 36.
4. Попова, О. І. Клінічна ефективність альтабору в комплексному лікуванні герпетичної інфекції порожнини рота. Український медичний
альманах 2013, 16 (1), 154 – 56.
5. Яковлєва, Л. В.; Євдокімова, О. С. Альтан – новий препарат для лікування виразкової хвороби шлунково-кишкового тракту. Вісник
фармації 1993, 1-2, 96 – 103.
6. Сербин, А. Г.; Яковлева, Л. В.; Хворост, О. П.; Гладух, Е. В.; Комиссаренко, Н. Ф.; Бондарь, В. С.; Болотов, В. В.; Шаламай, А. С. Альтан – новое оте-
чественное эффективное средство ранозаживляющего, противовоспалительного, антимикробного действия. Провизор 1998, 18, 40 – 41.
7. Бойчук, О. П. Застосування фітопрепарату “Альтан” в комплексному лікуванні хворих на гострі кишкові інфекції на тлі супутньої пато-
логії органів травлення. Галицький лікарський вісник 2004, 11 (2), 20 – 21.
8. Незгода, І. І.; Рикало, Н. А. (Вінницький національний медичний університет ім. М. І. Пирогова). Спосіб лікування гострих кишкових
інфекцій. Патент України 3317, 15.11.2004.
9. Рикало, Н. А. Ефективність фітопрепарату «Альтан» при експериментальному клебсієльозі. Експериментальна і клінічна медицина
2005, 2, 41 – 44.
10. Лапковський, Е. Й. Застосування альтану і дицетелу для корекції біохімічних та імунологічних показників при хронічному некамене-
вому холециститі. Клінічна та експериментальна патологія 2004, 4, 117 – 120.
11. Зупанец, И. А.; Герасименко, Е. В.; Шаламай, А. С.; Саенко, Т. В. Опыт применения препарата Альтабор в профилактике гриппа и острых
респираторных вирусных инфекций. Здоров’я України 2013, 20, 43 – 45.
12. Ivanov, S. A.; Nomura, K.; Malfanov, I. L.; Ptitsyn, L. R. Glutinoin, a novel antioxidative ellagitannin from Alnus glutinosa cones with glutinoic acid
dilactone moiety. Natural Product Research 2012, 26 (19), 1806 – 1816. https://doi.org/10.1080/14786419.2011.613387.
13. Кrutskikh, Т. V.; Shalamay, А. S. Validation of the method of quantitative determination of the amount elagotannins in tablets Altabor. Manage-
ment, economy and quality assurance in pharmacy 2015, 3, 25 – 29.
14. Gryszczyńska, A.; Dreger, M.; Piasecka, A.; Kachlicki, P.; Witaszak, N.; Sawikowska, A.; Ożarowski, M.; Opala, B.; Łowicki, Z.; Pietrowiak, A.; Miklaś, M.;
Mikołajczak Przemysław, Ł.; Wielgus, K. Qualitative and quantitative analyses of bioactive compounds from ex vitro Chamaenerion angustifolium (L.)
(Epilobium augustifolium) herb in different harvest times. Industrial Crops and Products 2018, 123, 208 – 220. https://doi.org/10.1016/j.ind-
crop.2018.06.010.
Journal of Organic and Pharmaceutical Chemistry. – 2021. – Vol. 19, Iss. 1 (73)
24
ISSN 2308-8303 (Print) ISSN 2518-1548 (Online)
15. Liang, X.; Jiang, Y.; Guo, Z.; Fang, S. Separation, UPLC-QTOF-MS/MS analysis, and antioxidant activity of hydrolyzable tannins from water caltrop
(Trapa quadrispinosa) pericarps. LWT 2020, 133, 110010. https://doi.org/10.1016/j.lwt.2020.110010.
16. Grace, M. H.; Warlick, C. W.; Neff, S. A.; Lila, M. A. Efficient preparative isolation and identification of walnut bioactive components using high-speed
counter-current chromatography and LC-ESI-IT-TOF-MS. Food Chem. 2014, 158, 229 – 238. https://doi.org/10.1016/j.foodchem.2014.02.117.
17. Regueiro, J.; Sánchez-González, C.; Vallverdú-Queralt, A.; Simal-Gándara, J.; Lamuela-Raventós, R.; Izquierdo-Pulido, M. Comprehensive identifi-
cation of walnut polyphenols by liquid chromatography coupled to linear ion trap–Orbitrap mass spectrometry. Food Chem. 2014, 152, 340 – 348.
https://doi.org/10.1016/j.foodchem.2013.11.158.
18. Jia, X.; Luo, H.; Xu, M.; Zhai, M.; Guo, Z.; Qiao, Y.; Wang, L. Dynamic Changes in Phenolics and Antioxidant Capacity during Pecan (Carya illinoinensis)
Kernel Ripening and Its Phenolics Profiles. Molecules 2018, 23 (2), 435. https://doi.org/10.3390/molecules23020435.
Received: 12. 05. 2020
Revised: 23. 01. 2021
Accepted: 15. 02. 2021
The work was supported by the PJSC SIC “Borshchahivskiy CPP” through the project “Development of methods
for determining the quantitative content of the components of the Altabor substance” (research period 2019).
|
| id | oai:ojs.journals.uran.ua:article-202081 |
| institution | Journal of Organic and Pharmaceutical Chemistry |
| issn | 2518-1548 |
| keywords_txt_mv | |
| language | English |
| last_indexed | 2026-08-25T01:01:50Z |
| publishDate | 2021 |
| publisher | National University of Pharmacy |
| record_format | ojs |
| resource_txt_mv | ophcjnupheduua/d1/3e3002be38746708c5840acf38ac9cd1.pdf |
| spelling | oai:ojs.journals.uran.ua:article-2020812026-08-24T15:33:48Z The determination of the phytochemical composition of the Altabor substance Визначення фітохімічного складу субстанції Альтабор Sheiko, S. Yu. Shalamay, A. S. gallotannins; ellagitannins; Altabor; chemical composition; structure; HPLC (high-performance liquid chromatography) галотаніни; елаготаніни; Альтабор; хімічний склад; структура; ВЕРХ (високоефективна рідинна хроматографія) The development of medicines based on alder cone extracts led to the introduction of Altan and Altabormedicines into medical practice. The technology of extraction cake from cones has made it possible to obtain extracts with different therapeutic properties.Aim. To develop an effective method for studying the qualitative composition of the Altabor substance anddetermine the quantitative content of its components.Results and discussion. The Altabor substance is a complex mixture of ellagitannins containing more than 70 components. The main components of the extract (or their isomers) were determined by mass spectrometry and by comparing the retention times with the literature data. Gallic, ellagic, valoneic acids dilactone were conclusively determined by adding reference standards of these acids to the extract. The substance contains the following compounds: 2,3-hexahydroxydiphenoyl-(α/β)-glucose tr1 = 0.55 min, tr2 = 0.89 min (α and β isomers), 4,6-O-[(S)-valeonyl]-D-glucose (isomer) tr = 0.64 min, gallic acid tr = 1.198 min, pedunculagin tr1 = 3.63 min, tr2 = 4.62 min(α and β isomers), proecoxin A (isomer) tr = 4.78 min, valoneic acid dilactone tr = 6.19 min, ellagic acid pentoside (isomer) tr = 7.07 min, ellagic acid tr = 7.335 min.Experimental part. The composition analysis was performed using an Agilent 1200 chromatograph with a UV detector, a G6140 mass detector, an Alltech 3300 light scattering detector (ELSD), as well as the Agilent ChemStation Rev.B.04.03 software. The molecular weights of the compounds in the extract were determined using the method of mass spectrometry of ESI-electrospray ionization. The determination of the components was performed using an ultraviolet detector at a wavelength of 280 nm. The column was Rapid Resolution HT Cartige, 4.6 × 30 mm, 1.8 μm, Zorbax SB-C18.Conclusions. A new effective method of analysis of the Altabor substance has been developed; it allowsdetermining the qualitative and quantitative content of its structural components. The method gives the possibility to control the process of obtaining the Altabor substance, study the dependence of its composition on the conditions of its obtaining, batch number, place, time of the natural raw material collection, and study the composition of other pharmaceutical substances, the plant raw material containing tannins. The advantage of the method isthe short time (up to 10 min) of analysis using high-performance liquid chromatography at high resolution.Key words: gallotannins; ellagitannins; Altabor; chemical composition; structure; HPLC (high-performance liquid chromatography) Розробка лікарських засобів на основі екстрактів суплідь вільхи вперше завершилась впровадженням у медичну практику препаратів Альтан та Альтабор. Особливості технології екстрагування підготовленого шроту суплідь дозволило отримувати екстракти з різними терапевтичними властивостями.Мета. Розробити ефективний метод вивчення якісного складу субстанції Альтабор із визначенням кількісного вмісту компонентів.Результати та їх обговорення. Субстанція Альтабор становить собою складну багатокомпонентну суміш елаготанінів, що містить більше 70 компонентів. Основні компоненти екстракту визначено за допомогою мас-спектрометрії, а також шляхом порівняння часів утримування з літературними даними. Галову, елагову, валонову кислоти визначено остаточно шляхом додавання в екстракт стандартів цих кислот. До його складу входять ідентифіковані сполуки: 2,3-гексагідроксидифеноїл-(α/β)-глюкоза tr1 = 0,55 хв, tr2 = 0,89 хв (α та β ізомери), 4,6-O-[(S)-валеоніл]-D-глюкоза (ізомер) tr = 0,64 хв, галова кислота tr = 1,198 хв, педункулагін tr1 = 3,63 хв, tr2 = 4,62 хв (α та β ізомери), праекоксін А (ізомер) tr = 4,78 хв, дилактон валонової кислоти tr = 6,19 хв, пентозид елагової кислоти (ізомер) tr = 7,07 хв, елагова кислота tr = 7,335 хв.Експериментальна частина. Аналіз складу проводили за допомогою Agilent 1200 хроматографа з УФ-детектором, мас-детектором G6140 та детектором світлорозсіювання (ELSD) Alltech 3300. Програмне забезпечення Agilent ChemStation Rev.B.04.03. Молекулярні маси сполук, які входять до складу екстракту, визначено за допомогою методу мас-спектрометрії іонізацією розпиленням в електричному полі (ESI-electrospray ionization). Визначення компонентів проводили із застосуванням ультрафіолетового детектора за довжини хвилі λ 280 нм. Колонка: Rapid Resolution HT Cartige 4,6 × 30 мм, 1,8 мкм, Zorbax SB-C18.Висновки. Розроблено новий ефективний метод аналізу фармсубстанції Альтабор, який дозволяєвивчати її якісний склад та визначати кількісний вміст компонентів. Метод дозволяє контролювати процес виробництва субстанції Альтабор, вивчати залежність її складу від умов отримання, номеру партії, місця, часу збору природної сировини, вивчати склад інших фармсубстанцій, природної сировини, що містить таніни. Перевага методу – короткий час (до 10 хвилин) проведення аналізу за допомогою високоефективної рідинної хроматографії за високої роздільної здатності.Ключові слова: галотаніни; елаготаніни; Альтабор; хімічний склад; структура; ВЕРХ (високоефективна рідинна хроматографія) National University of Pharmacy 2021-03-15 Article Article application/pdf https://ophcj.nuph.edu.ua/article/view/202081 10.24959/ophcj.21.202081 Journal of Organic and Pharmaceutical Chemistry; Vol. 19 No. 1(73) (2021); 16-24 Журнал органической и фармацевтической химии; Том 19 № 1(73) (2021); 16-24 Журнал органічної та фармацевтичної хімії; Том 19 № 1(73) (2021); 16-24 2518-1548 2308-8303 en https://ophcj.nuph.edu.ua/article/view/202081/226697 Copyright (c) 2021 S. Yu. Sheiko, A. S. Shalamay http://creativecommons.org/licenses/by/4.0 |
| spellingShingle | галотаніни елаготаніни Альтабор хімічний склад структура ВЕРХ (високоефективна рідинна хроматографія) Sheiko, S. Yu. Shalamay, A. S. Визначення фітохімічного складу субстанції Альтабор |
| title | Визначення фітохімічного складу субстанції Альтабор |
| title_alt | The determination of the phytochemical composition of the Altabor substance |
| title_full | Визначення фітохімічного складу субстанції Альтабор |
| title_fullStr | Визначення фітохімічного складу субстанції Альтабор |
| title_full_unstemmed | Визначення фітохімічного складу субстанції Альтабор |
| title_short | Визначення фітохімічного складу субстанції Альтабор |
| title_sort | визначення фітохімічного складу субстанції альтабор |
| topic | галотаніни елаготаніни Альтабор хімічний склад структура ВЕРХ (високоефективна рідинна хроматографія) |
| topic_facet | gallotannins ellagitannins Altabor chemical composition structure HPLC (high-performance liquid chromatography) галотаніни елаготаніни Альтабор хімічний склад структура ВЕРХ (високоефективна рідинна хроматографія) |
| url | https://ophcj.nuph.edu.ua/article/view/202081 |
| work_keys_str_mv | AT sheikosyu thedeterminationofthephytochemicalcompositionofthealtaborsubstance AT shalamayas thedeterminationofthephytochemicalcompositionofthealtaborsubstance AT sheikosyu viznačennâfítohímíčnogoskladusubstancííalʹtabor AT shalamayas viznačennâfítohímíčnogoskladusubstancííalʹtabor AT sheikosyu determinationofthephytochemicalcompositionofthealtaborsubstance AT shalamayas determinationofthephytochemicalcompositionofthealtaborsubstance |