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 VOLUME Vol.05 Issue01 2025 

PAGE NO. 23-27 

DOI 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 

 

https://doi.org/10.37547/ajbspi/Volume05Issue01-06
https://doi.org/10.37547/ajbspi/Volume05Issue01-06
https://doi.org/10.37547/ajbspi/Volume05Issue01-06
https://doi.org/10.37547/ajbspi/Volume05Issue01-06


American Journal of Applied Science and Technology 24 https://theusajournals.com/index.php/ajast 

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 



American Journal of Applied Science and Technology 25 https://theusajournals.com/index.php/ajast 

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 = 1.0000 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 solution 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 



American Journal of Applied Science and Technology 26 https://theusajournals.com/index.php/ajast 

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 



American Journal of Applied Science and Technology 27 https://theusajournals.com/index.php/ajast 

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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Ginzburg Z.K. Biological activity of substances of 
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Lapayeva L.G., Morozova E.V. Methods of analysis of 
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Kaur, N., Singh, R. Phytochemical analysis and 
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Yin, J., Zhu, X. Advances in research on Acorus calamus: 
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* Pharmaceutical Bulletin*, 2020, 4 (2), 30-35.  

 

 


