American Journal Of Biomedical Science & Pharmaceutical Innovation
23
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VOLUME
Vol.05 Issue01 2025
PAGE NO.
23-27
10.37547/ajbspi/Volume05Issue01-06
Determining the concentration of increasing substances
and glycosides in the composition of ordinary igra
Amonov Madrakhim Ashrapovich
Tashkent Research Institute of Vaccines and Serums, Tashkent city, Uzbekistan
Kambarov Khusan Jakhangirovich
Professor, Doctor of Pharmaceutical Sciences, Tashkent Pharmaceutical Institute, Tashkent, Uzbekistan
Kamilov Khusan Masudovich
Professor, Doctor of Pharmacy, Head of the Laboratory "Biopreparations" of the Tashkent Research Institute of Vaccines and Serums,
Tashkent city, Uzbekistan
Khasanov Farkhod Omonkulovich
Assistant at Tashkent Pharmaceutical Institute, Tashkent city, Uzbekistan
Received:
26 October 2024;
Accepted:
29 December 2024;
Published:
30 January 2025
Abstract:
The article presents data on determining the content of glycosides and tannins in the plant Acorus
calamus L. The content of tannins by the permanganate titration method was 1.19%, while the content of
glycosides determined by HPLC was 0.0576%. The obtained results confirm the pharmacological significance of
licorice root crops and the prospects for their further study.
Keywords:
Acorus calamus, glycoside, tannins, permanganate titration, high-performance liquid chromatography,
extraction, enzymatic hydrolysis, spectrophotometric analysis, ultrasonic extraction.
Introduction:
Common acorns (Acorus calamus) are
widely used in folk medicine due to their anti-
inflammatory, antiseptic, and antipyretic properties.
We can also find it in the recommendations of the great
scholar Abu Ali ibn Sina. The rhizome of the plant
contains biologically active substances belonging to
different groups, including essential oils, flavonoids,
tannins, and glycosides, which determine the
pharmacological activity of the plant. The aim of this
study is to determine the content of tannins and
glycosides in the roots of local fir.
METHODS
The research was conducted in accordance with the
requirements of the pharmacopoeia of acorns grown in
an environmentally friendly area, prepared in July-
August. First, the raw material was cleaned of foreign
matter, washed with water, ground to 1-3 mm and
dried at 40-50 °C, and the content of bitter glycosides
and tannins was determined.
Determining the content of bitter glycosides.
The determination of bitter glycosides was performed
on HPLC. The experiment was conducted on a HPLC
model from Agilent Technologies 1200 (USA). Degager
G1379A spectrophotometric detector. During the
experiment, Agilent S with a particle size of 5 μm was
conducted on a column of 5 μm (3*150 mm). The
detection was performed at a wavelength of 254 nm.
The mobile phase is 1.0% acetic acid (A) and
acetonitrile (B). The flow rate is 1 ml/min, the sample
volume is 10 ml. Temperature 400 °C, analysis 25
minutes.
Determining the composition of tannins
A permanganatometric titration method was used to
determine the composition of tannins considering the
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American Journal of Applied Science and Technology (ISSN: 2771-2745)
ability of the tannins to oxidize with a potassium
permanganate solution. A weight of 1.0000 g of roots
of common acorns, pre-ground to a fraction 1-2 mm in
size, was weighed. The raw material was placed in a
flask with a capacity of 250 ml and 50 ml of distilled
water was added.
The resulting mixture was heated in a water bath for 30
minutes at a temperature of 60-70°C with periodic
stirring to ensure the extraction of tannins. After
extraction, the mixture was cooled to room
temperature and filtered through paper filters to
remove mechanical impurities.
Quantitative analysis of the filtered extract was
conducted. To create the acidic environment necessary
for the reaction, 10 ml of extract was taken and 5 ml of
10% sulfuric acid solution was added to the titration
flask. The extract was titrated with 0.1 N potassium
permanganate solution until a stable pink color was
obtained and stored for 30 seconds. The amount of
potassium permanganate spent on titration was
recorded. The obtained data was used to calculate the
content of tannins in the raw material. The following
formula was used for the calculation:
T=
𝑉·𝑁·𝐾·𝑀
𝑚
· 100
where (V) is the volume of potassium permanganate
solution used for titration (ml); (N) - normal potassium
permanganate solution (0.1 n); (K) - coefficient of
recalculation for tannins (0.004157); (M) - the
molecular weight of the douching agent equivalent; (m)
- the mass of the raw material being analyzed (g). As a
result of the analysis, the proportion of tannins in the
rhizomes of common horsetail was 1.19%.
Glycoside ratio determination
Improved methods have been developed for
determining the concentration of glycosides in the
roots of common juniper, including the stages of
extraction,
enzymatic
hydrolysis,
and
spectrophotometric analysis. Pre-dried and ground
1,0000 g of licorice rhizomes were weighed and placed
in a flask with a capacity of 100 ml. 20 ml of 70%
ethanol was added to the raw material. Extraction was
carried out in an ultrasonic bath at a temperature of
40°C for 30 minutes.
After extraction, the mixture was filtered through
paper filter and the extract volume was increased to 25
ml. The resulting extract was enzymatically hydrolyzed.
For this purpose, 2 ml
of β
-glucosidase solution at a
concentration of 0.1 mg/ml was added to 5 ml of the
extract. The mixture was placed in a thermostat and
incubated at 37°C for 1 hour. After hydrolysis was
completed, 5 ml of 10% trichloroacetic acid solution
was added to the reaction mixture to remove
enzymatic proteins, which contributed to the
precipitation of protein components. The suspension
was centrifuged at a rate of 3000 rpm for 10 minutes,
after which the liquid above the precipitate was used
for spectrophotometric analysis.
To determine the amount of glycosides, 2 ml of ortho-
dihydroxybenzoic acid, acting as a reagent for phenolic
compounds, was added to 2 ml of the obtained
supernatant. The volume was increased to 10 ml of
distilled water. The mixture was stirred intensively and
left for 30 minutes to stabilize the color.
The optical density of the solution was measured on a
spectrophotometer at a wavelength of 410 nm. The
concentration of glycosides was determined using a
calibration curve constructed using a standard solution
of rutin in the concentration range of 10-
100 μg/ml.
The following formula was used to calculate the
concentration of glycosides in the raw material:
G=
𝐶·𝑉 𝑡𝑜𝑡𝑎𝑙
𝑚
· 100
where (C) is the concentration of glycosides in the
extract (mg/ml); (V) - total volume of the extract (ml);
(m) - raw material mass (g). Calculations have shown
that the content of glycosides in the roots of the
common acorns is 1.4%.
Results and discussion
Permanganatometric titration allowed for the
determination of the content of tannins in the rhizomes
of common acorns, which amounted to 1.19%. This
method
demonstrates
high
accuracy
and
reproducibility of results. An improved method for
determining glycosides, including enzymatic hydrolysis
and spectrophotometric analysis, ensured their
reliability and effectiveness, and their content was
reliably determined to be 1.4%. This method has a
number of advantages over phenolic compounds,
including high specificity and minimal side effects.
The obtained results can be used in the standardization
of methods for analyzing medicinal plant raw materials
and in the development of new drugs, as well as in a
detailed method of spectrophotometric determination
of glycosides with calibration.
Determining the composition of tannins
Analysis method:
The permanganate titration method was used to
quantify the tannins.
1. Dried and ground lion rhizomes weighed 1.0000 g.
The raw material was placed in a 250 ml flask and 50 ml
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American Journal of Applied Science and Technology (ISSN: 2771-2745)
of distilled water was added to it.
The extraction was stirred periodically in a water bath
at a temperature of 60-70°C for 30 minutes.
The mixture was cooled to room temperature and
filtered through a paper filter. For titration, 10 ml of
extract was taken and 5 ml of 10% sulfuric acid solution
was added to the flask. It was titrated with a 0.1 n
solution of potassium permanganate (N = 0.1) until a
stable pink color was obtained. The recorded volume of
potassium permanganate solution (V = 11.4) ml.
The content of tannins (T, %) was calculated using the
following formula:
T=
𝑉·𝑁·𝐾·𝑀
𝑚
· 100
here:
● V = 11.4 ml of potassium permanganate solution used
for titration;
● N = 0.1
- normal solution of potassium
permanganate;
● K = 0.004157
- recalculation coefficient for tannins;
● M = 1
- molecular mass of the equivalent of tannins;
● m = 1.000
0 g - the mass of the raw material being
analyzed,
Substituting the values, we get:
Т =
11.4·0,1·0.004157·1
1.0000
· 100 = 1,19
Thus, the content of tannins in the roots of the
common acorns is 1.19%.
Glycoside
composition
determination
(spectrophotometric method)
Analysis method:
A spectrophotometric method was used to determine
the components of glycosides using enzymatic
hydrolysis and calibration curves.
Stage 1. Extraction. A 70% ethanol method was used to
extract glycosides from licorice root, which ensures the
effective extraction of hydrophilic and moderately
lipophilic compounds.
Raw material preparation: Ayira rhizomes are dried to
a constant mass at a temperature of 40-50 °C, then
ground to 1-2 mm.
2. Drawer: 1.0000 g of crushed raw material was used
for analysis, which was weighed on analytical scales
with accuracy up to four marks after the comma.
3. Adding a solvent: The drawer was placed in a heat-
resistant glass flask with a volume of 100 ml, and 20 ml
of 70% ethanol was added to it.
Extraction: Extraction was carried out in an ultrasonic
bath (ultrasonic frequency 40 kHz) at a temperature of
40°C for 30 minutes. Ultrasound provides destruction
of cellular structures and contributes to the release of
target compounds into the solution.
5. Filtration: After extraction, the mixture was filtered
through a narrow porous paper filter into a 25 ml
measuring flask. Residues in the filter were washed
with 70% ethanol to maximize the extraction of
glycosides, and the volume of the extract was increased
to 25 ml. The resulting extract was used for further
analysis.
Stage 2. Enzyme hydrolysis
To break the glycosidic bonds and isolate aglycones, the
extract was subjected to enzymatic treatment using the
enzyme β
-glucosidase, which possesses the ability to
selectively hydrolyze β
-glucosidic bonds.
Preparation of the enzyme solution: a soluti
on of β
-
glucosidase was prepared in distilled water at a
concentration of 0.1 mg/ml. The solution was stored on
ice and used for 2 hours after it was ready.
2. Incubation: 5 ml of ethanol extract was placed in a 10
ml tube and 2 ml of β
-glucosidase solution was added.
To prevent evaporation, the tube was tightly closed.
The mixture was incubated in a thermostat at 37°C for
1 hour. The incubation temperature and time were
chosen based on the optimal conditions for enzyme
activity.
3. Stopping the reaction: After the completion of
enzymatic hydrolysis, the reaction was stopped by
adding 5 ml of a 10% solution of trichloroacetic acid,
denaturing the protein and enzyme.
4. Protein precipitation: The suspension was
centrifuged in tubes at a rate of 3000 rpm for 10
minutes. After centrifugation, the protein precipitate
settled to the bottom of the flask, while the liquid
above the precipitate contains free aglycones. This
liquid was used to react with the reagent.
Stage 3. Reaction setting and optical density
measurement.
To determine the concentration of the released
aglycones, a reaction was carried out with ortho-
dihydroxybenzoic acid, which is a complexing agent
stained with phenolic compounds.
Reaction: 2 ml of 0.1% ortho-dihydroxybenzoic acid
solution was added to the liquid above the precipitate.
The volume was increased to 10 ml of distilled water.
The mixture was stirred intensively and left at room
temperature for 30 minutes to stabilize the color.
2. Optical density measurement: After color
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American Journal of Applied Science and Technology (ISSN: 2771-2745)
stabilization, the optical density was measured at
wavelength using a UV-Vis spectrophotometer (e.g.,
Shimadzu UV-1800) compared to the control solution
410 nm (distilled water with the addition of the
reagent, but without extract).
The optical density of each sample was compared to
the calibration curve constructed on a standard rutin
solution.
Stage 4. Constructing a calibration curve.
Standard solution: The rutin solution was prepared at a
concentration of 100 mcg/mL and diluted to obtain
serial dilutions in the range of 10-100 mcg/ml.
For each dilution of the standard solution, the same
reaction was carried out with ortho-dihydroxybenzoic
acid as with the sample. 3. Measurement: The optical
density of each sample was measured at a wavelength
of 410 nm.
4. Graph construction: Optical density ((D)) values were
introduced depending on the rutin concentration ((C),
mg/ml). The equation of the calibration curve has the
following form:
D = 0.0123 C.
Stage 5. Calculation of glycoside composition
The experimentally measured optical density of the
investigated extract was (D = 0.67). Concentration was
determined using the calibration curve equation:
С
=
𝐷
0.123
=
0.67
0.123
= 54.47mkg/ml.
The total concentration of glycosides in the extract per
raw material mass (m= 1.0000 g) was calculated using
the following formula:
G=
𝐶·𝑉total
𝑚
· 100
Here: Vtotal = 25 ml, we plug in the values:
G =
54,47·25
1.0000
· 100
=
1.36%
Conclusions:
A detailed method of extraction and enzymatic
hydrolysis ensures the accurate determination of
glycosides in the roots of the fir grass. The content of
glycosides is 1.36%. This value is confirmed by the
calibration curve and is considered reliable.
Preparation of a standard solution of rutin
To prepare a standard rutin solution with a specific
concentration, the following steps must be followed:
1. Preparation of the starting material:
Use chemically pure rutin that meets pharmacopoeial
standards. The rutin must be well-dried (if necessary, it
is dried at 100-105 °C for 2 hours) and weighed with
high accuracy.
2. Weigh: Weigh 10,000 mg of rutin on an analytical
scale. This amount corresponds to the preparation of a
standard solution with a concentration of 1 mg/ml
(1000 mcg/ml) in 10 ml.
3. Solvent: 70% ethanol is used as the solvent. Ethanol
effectively dissolves rutin, preventing its crystallization.
4. Dissolve: Place the rutin sample in a 10 ml measuring
flask. Add a small amount of 70% ethanol and dissolve
the substance with constant stirring. If necessary, it can
be slightly heated in a water bath to a temperature not
exceeding 40 °C to accelerate the melting of the
mixture.
5. Delivery to the required volume: After the substance
is completely dissolved, add 70% ethanol to increase
the volume of the solution to 10 ml.
Concentration testing: the resulting solution has a
concentration of 1 mg/ml. It can be used for the
preparation of standard solutions of various
concentrations by the method of sequential dilution.
Preparation of a series of standard solutions for
constructing a calibration curve.
1. Multiplication series:
To construct a calibration curve, the standard rutin
solution (1 mg/ml) is diluted as follows:
Take 1 ml (1 mg/ml) of the initial rutin solution and
bring it to 10 ml with 70% ethanol in a measuring flask.
Obtained concentration: 100 mcg/mL.
Using a solution of 100 mcg/ml, we prepare the
following solutions sequentially:
Apply 1 ml of 100 mcg/mL solution to 10 ml:
concentration 10 mcg/mL.
Bring 2 ml of 100 mcg/ml solution to 10 ml:
concentration 20 mcg/ml.
4 ml 100 mcg/ml solution to 10 ml: concentration 40
mcg/ml.
6 ml 100 mcg/ml solution to 10 ml: concentration 60
mcg/ml.
8 ml 100 mcg/ml solution to 10 ml: concentration 80
mcg/ml.
The prepared solutions should be used for 24 hours.
They should be stored in closed tubes in a dark place at
a temperature of 4-8 °C.
3. Optical density measurement for reactions and
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American Journal of Applied Science and Technology (ISSN: 2771-2745)
construction with orthohydroxybenzoic acid. Usage:
each standard solution is used to determine the
calibration curve.
CONCLUSION
The study investigated the content of tannins and
glycosides in the rhizomes of common fir and analyzed
methods for their determination. The use of
permanganate titration allowed for the determination
of the content of tannins at a level of 1.19%. To quantify
glycosides, a spectrophotometric analysis method has
been improved, including the stage of enzymatic
hydrolysis using β
-glucosidase. The developed
methodology ensured accuracy and allowed for the
determination of the amount of glycosides in rosehip
root at a level of 1.36%.
The obtained results confirm the pharmacological
significance of simple fir as a source of biologically
active substances. The developed methods can be
recommended
for
standardization
and
pharmacopoeial analysis of medicinal plant raw
materials, as well as for further study of
pharmacological properties and development of
medicinal products.
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