Volume 04 Issue 09-2024
58
International Journal of Medical Sciences And Clinical Research
(ISSN
–
2771-2265)
VOLUME
04
ISSUE
09
P
AGES
:
58-66
OCLC
–
1121105677
Publisher:
Oscar Publishing Services
Servi
ABSTRACT
Bioactive groups were identified and methods for their identification and determination in the herbal preparation
Sedavit, tablets, were developed. Methods were developed for high-performance liquid chromatography (HPLC) for
the identification of polyphenolic compounds and the quantitative determination of pyridoxine and nicotinamide; gas
chromatography (GC) for the determination of isovaleric acid, spectrophotometry (SP) for the quantitative
determination of flavonoids.
KEYWORDS
Herbal medicines, Sedavit, identification, quantitative determination, control methods.
INTRODUCTION
Herbal medicines consist of a complex of substances
belonging to several chemical groups and under the
influence of technological processes or internal
enzymatic reactions, chemical transformations of
these substances can occur. The task of standardizing
medicinal plant raw materials and drugs based on them
Research Article
DEVELOPMENT OF METHODS FOR IDENTIFICATION AND
DETERMINATION OF BIOLOGICALLY ACTIVE SUBSTANCES IN SEDAVIT
HERBAL TABLET PRODUCTS
Submission Date:
Sep 20, 2024,
Accepted Date:
Sep 25, 2024,
Published Date:
Sep 30, 2024
Crossref doi:
https://doi.org/10.37547/ijmscr/Volume04Issue09-09
Salomov Shokhabbos Nozimjon ugli
Student of Andijan State Medical Institute, Uzbekistan
Djalalova Ozoda Kasimjanovna
PhD. Department of Pathophisiology, Andijan State Medical Institute, Uzbekistan
Journal
Website:
https://theusajournals.
com/index.php/ijmscr
Copyright:
Original
content from this work
may be used under the
terms of the creative
commons
attributes
4.0 licence.
Volume 04 Issue 09-2024
59
International Journal of Medical Sciences And Clinical Research
(ISSN
–
2771-2265)
VOLUME
04
ISSUE
09
P
AGES
:
58-66
OCLC
–
1121105677
Publisher:
Oscar Publishing Services
Servi
is often complicated by the lack of accurate control
indicators and the existing indicators are not accepted
for testing qualitative and quantitative properties. The
main goal of our work is to develop a method for
standardizing finished pharmaceutical products (PPF)
on a plant basis, ensuring their effectiveness and safety
for consumers, ensuring the development of
appropriate technical parameters and quality control
methods, among other things, for the identification of
counterfeit drugs [1, 2]. Standardization includes the
following interrelated stages of pharmaceutical
product development:
–
Select individual compounds, biologically active
compounds (BAC) or markers to assess the quality of
FMPs;
–
select methods and develop techniques for the
identification and/or quantification of individual
compounds, BAS groups or markers in FMPs;
–
selection and substantiation of standardization
criteria taking into account the requirements of
pharmacopoeial monographs, data from scientific
literature
and
characteristics
of
production
technology.
Let's look at each of these moments using the example
of the development process of a finished herbal
product, Sedavit tablets.
METHODS
The object of our study is the drug Sedavit, in tablet
form, whose active ingredient is a concentrated
extract containing 5 plant ingredients (valerian
rhizome, hawthorn fruit, St. John's wort, peppermint
leaves, hop cones), vitamins - nicotinamide (vitamin P)
and pyridoxine hydrochloride (vitamin B6).
The study used gas chromatography (GC) and high-
performance liquid chromatography (HPLC). GC
studies were performed
on an Agilent 6890 N” gas
chromatograph with a flame ionization detector
(Agilent, USA). For HPLC studies, an Agilent 1200 liquid
chromatograph with a diode array detector (Agilent,
USA) was used. Quantitative determination of total
flavonoids
was
performed
on
a Cary
100
spectrophotometer (Varian, Australia). The study used
standards including chlorogenic, caffeic, ferulic and
rosmarinic acids (Fluka), rutin, hypersaccharide,
luteolin, quercetin, apigenin (Fluka). The reagents used
for the study complied with the requirements of the
European Pharmacopoeia (EPh) and the State
Pharmacopoeia of Uzbekistan (SPU). The reaction
solutions
were
prepared
according
to
the
requirements of EP/SPU [3, 4].
RESULTS AND DISCUSSION
Isolation of individual compounds, groups of bioactive
substances or markers. Each plant component of the
complex extract contains a variety of bioactive
substances, the composition and proportion of which
Volume 04 Issue 09-2024
60
International Journal of Medical Sciences And Clinical Research
(ISSN
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2771-2265)
VOLUME
04
ISSUE
09
P
AGES
:
58-66
OCLC
–
1121105677
Publisher:
Oscar Publishing Services
Servi
may vary depending on the geographical and climatic
conditions of growth and cultivation, harvesting,
drying, etc. The complex extract is obtained by
simultaneous extraction of a mixture of five plant
materials. This complicates the development of a
control strategy that includes the qualitative or
quantitative determination of the bioactive substances
or markers inherent in each medicinal plant material
(MPM), since the extract will contain both substances
common to different MPMs and substances
characteristic of one MPM. In this regard, we
conducted a literature review on BAS of the studied
raw materials [5
–
7], thanks to which we selected
groups of substances whose presence and content
could be used for identification and quantitative
determination (evaluation results are presented in the
table).
Table 1. Biologically active substances of the studied types of raw materials
Raw materials
Compounds
Peppermint leaves
Essential oil (from 2.40 to 3.75%). Essential
oil basically consists of menthol (41-65%),
menthone and menthyl acetate. Carotene,
flavonoids:
apigenin,
hesperidin,
rutin,
chlorogenic, rosmarinic, ursolic and oleanolic
acids; microelements: copper, manganese,
strontium, etc.
St. John’s wort herb
Flavonoids: hypericin, hyperoside, rutin,
quercitrin, quercetin, chlorogenic, caffeic
acids,
tannins,
coumarins,
carotenoids,
vitamins C and PP
Hawthorn berries
Flavonoids: quercetin, hyperoside, rutin, etc.;
chlorogenic,
caffeic
acids,
glycosides,
carotenoids, tannins, fatty oils, saponins,
pectin substances, etc.
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Rhizomes with roots of valerian
Essential oil (from 0.5 to 2%), the main part of
which is valerianic-borneol ester, various
organic acids: valerianic, isovaleric, formic,
acetic, etc., valepotriates, alkaloids: valerine,
chatinine, etc.
Hop cones
Bitter
substances
humulone,
lupulone,
humulenic
acid,
humulinone
isomers,
essential oil, flavonoids rutin, quercitrin,
tannins, etc.
According to literature data [5
–
7], the BAS
composition of four MPMs (hawthorn berries, St.
John's wort, peppermint leaves, hop cones) included
phenolic compounds, which were selected based on
their qualitative and quantitative properties. To
demonstrate the presence of valerian rhizomes in
MPMs, isovaleric acid was chosen as a marker.
Selection of criteria and methods for the identification
and/or quantification of individual compounds, groups
of biologically active substances or markers in the
finished medicinal product. When choosing a control
method, it is necessary to take into account the
selectivity, sensitivity of the method, and suitability for
quality control in the medicinal plant raw material -
extract - concentrate - finished product chain. Highly
selective chromatographic control methods best meet
these requirements and are suitable for the
standardization of herbal preparations [8]. Liquid
chromatography is proposed for the identification and
quantification
of
the
synthetic
components
nicotinamide (vitamin P) and pyridoxine hydrochloride
(vitamin B6). The conditions for conducting the
analysis allow for simultaneous identification and
quantitative determination. Liquid chromatography
was chosen for the determination of phenolic
compounds
in
pharmaceutical
products.
The
substances were identified by comparing the retention
times of the peaks in the chromatograms of the test
solutions with those of the standards. During the
development of the method, samples of chlorogenic,
caffeic,
ferulic
and
rosmarinic
acids,
rutin,
hypersaccharide, luteolin, quercetin and apigenin were
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used as standards. For routine control, chlorogenic,
rosmarinic, caffeic and quercetin acids were chosen as
the most specific representatives of the biologically
active substances of the plant components contained
in the drug. Chromatography was performed on a
liquid chromatograph with a UV detector using a C18
chromatographic column; mobile phase A: 0.6 g/l
sodium dihydrogen phosphate monohydrate solution
with pH 2.5 (using phosphoric acid), mobile phase B:
acetonitrile; mobile phase flow rate - 1.0 ml/min;
detection at 330 nm. Typical chromatograms of the
comparison solution and the test solution are shown in
Figures 1.1 and 1.2. Gas chromatography was used to
determine isovaleric acid in the rhizomes and roots of
Valerian in FPP. The determination was carried out by
comparing the retention time of the main peak in the
chromatogram of the test solution with the retention
time of the isovaleric acid peak, a substance
characteristic of the rhizome and root of valerian. The
chromatography was carried out on a gas
chromatograph with a flame ionization detector, using
a 60 m capillary column with an internal diameter of
0.53 mm, the stationary phase was macrogol 20000 P
with a layer thickness of 1 μm; the carrier gas was
helium for chromatography P, the carrier gas flow rate
was 5 ml/min, the flow distribution was 1:1. Typical
chromatograms of isovaleric acid comparison solution
and Sedavit tablet test solution are shown in Figures 2.1
and 2.2.
Figure 1.1 Typical chromatogram of the reference solution obtained during the identification of
phenolic compounds
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Figure 1.2 Typical chromatogram of the test solution of Sedavit, tablets, obtained during the
identification of phenolic compounds
When selecting quantitative determination criteria, the
content of biologically active substances in plant raw
materials, semi-finished products and technological
features should be taken into account. The
requirements should reflect the actual level of
biologically active substances, which characterizes the
effectiveness of the
Figure 2.1 Typical chromatogram of the comparison solution of isovaleric acid obtained during
its identification
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Figure 2.2 Typical chromatogram of the test solution of Sedavit, tablets, obtained during the
identification of isovaleric acid
finished medicinal product. In this case, if the finished
medicinal product contains a negligible amount of
biologically active substances or markers, the
specification should only include identification
requirements and during quantitative testing the
amount of biologically active substances should be
determined; in this case, tests can be carried out, for
example, by spectrophotometric methods. Sedavit
tablets from the UK contain a complex extract
consisting of five medicinal plant raw materials. Its
therapeutic effect is determined by a complex of
biologically active substances isolated from plant raw
materials. In this regard, when quantitatively
Figure 3 Differential electronic absorption spectra of the aluminum chloride complex with
flavonoids of the studied Sedavit solution, tablets (1) and standard rutin solution (2)
analyzing active substances, it is unreasonable to
determine
each
component
individually
by
chromatography and then calculate the total peak
area. For these purposes, it is sufficient to use
spectrophotometric methods to determine the total
optical density of the components determined under
the analytical conditions [9]. When choosing a
standard sample for recalculating the active substance
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content, we took into account its availability for
routine control, the inclusion of the selected substance
in the determined compound group and the need to
use conversion factors [9]. French rutin was chosen as
the standard, which under analytical conditions gave a
maximum absorbance at 415 nm and did not show a
reliable difference in spectrum compared to the
indicator of the analyzed solution. For routine control,
it is proposed to use the absorption index value of rutin
with aluminium chloride, which simplifies and reduces
the cost of the method for quantitative determination
of flavonoids, a typical group for medicinal plant raw
materials used. The optical density of the test solution
was determined after the formation of a colored
complex of flavonoids extracted with ethyl acetate and
aluminium chloride. The initial solution (ethyl acetate
extract) without adding aluminium chloride reagent
was used as a compensation solution, which avoids the
influence of the accompanying substances extracted
with ethyl acetate on the analytical results. In parallel,
the optical density of the rutin standard sample
solution with aluminium chloride was measured using
the initial rutin standard solution without adding
aluminium chloride reagent as a compensation
solution. Typical differential absorption spectra of the
aluminium chloride complex with flavonoids of the test
sample (complex extract) and the rutin standard are
shown in Figure 3.
CONCLUSION
As a result of this work, a herbal medicine
standardization algorithm was proposed using Sedavit
tablets as an example, including conditions for
selecting bioactive groups and markers, identification
and quantitative determination methods, qualitative
and quantitative methods, and quantitative criteria for
drug evaluation.
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