Determination of ICARIIN in Traditional Chinese Medicine

Preparation by HPLC

Ying Wang

, Haitao Gong

, Cuifang Gao

, Yun Zhou

, Yonghe Wang

and Yanqun Wang

1,*

Shandong Drug and Food Vocational College, Zibo, Shandong, 255011, China

Jinan Zhangqiu District Hospital of TCM, Jinan, Shandong, 250200, China

Keywords: HPLC, Icariin.

Abstract: A method for the determination of ICARIIN in traditional Chinese medicine by High-paerformance liquid

chromatography was established, in which octadecyl Silane Bonded Silica Gel was used as Filler, acetonitrile-

water (30:70) as mobile phase, the detection wavelength was 270 nm, the flow rate was 1μl /min, the injection

volume was 10μl. Column Temperature is 40℃. The average recovery was 99.97%. LINEAR

CORRELATION COEFFICIENT r=0.9999, precision and repeatability RSD were 0.35% and 0.70%

respectively.

1 INTRODUCTION

The traditional Chinese medicine preparation

Wenshen Huazhuo Capsule (formerly known as

Jiangzhi Capsule) is a well-known traditional

Chinese medicine prescription in Zhangqiu District

Traditional Chinese Medicine Hospital. It is based on

many years of clinical practice experience and a

combination of traditional Chinese medicine theories.

The prescription embodies the guiding ideology of

holistic concept and differentiation and treatment of

traditional Chinese medicine. It has the effects of

warming the kidney and resolving turbidity,

promoting blood circulation and channeling. It can be

used for hyperlipidemia caused by deficiency of

kidney yang and blood turbidity and blood stasis. The

curative effect is remarkable. The prescription

contains medicinal flavors such as roasted

Epimedium, Hawthorn, Mistletoe, Polygonatum

odoratum, which have the effects of lowering blood

fat and nourishing the kidney and removing blood

stasis. In order to give full play to the advantages of

traditional Chinese medicine in lowering lipids and

facilitating the consumption of patients, the

preparations for medical institutions have been made,

and the preparation of traditional Chinese medicine

preparations by medical institutions using traditional

techniques has been completed. While exploring and

improving its process and formulating process

standards, a method for determining the content of

icariin is established as the main control index of the

internal control agent standard. This article uses

HPLC to determine its content.

2 INSTRUMENT AND TEST

MEDICINE

Instrument: Shimadzu SPD-20AT high performance

liquid chromatograph; Ajilent C18 column

250×4.6mm 5μm

Icariin reference substance: the batch number is

110737-201516, the content is 94.2%, and it is

purchased from the China Institute for Food and Drug

Control.

Reagents: German Merck chromatographic pure

acetonitrile, analytical pure ethanol, Wahaha pure

water.

3 METHODS AND RESULTS

3.1 Chromatographic Conditions and

System Applicability Test

It can use octadecylsilane-bonded silica gel as filler;

use acetonitrile-water (30:70) as mobile phase; SPD-

20A ultraviolet detector. Detection wavelength is

270nm, flow rate: 1.0ml/min; injection volume: 10μl;

872

Wang, Y., Gong, H., Gao, C., Zhou, Y., Wang, Y. and Wang, Y.

Determination of ICARIIN in Traditional Chinese Medicine Preparation by HPLC.

DOI: 10.5220/0011311800003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 872-877

ISBN: 978-989-758-595-1

Column temperature: 40°C. The number of

theoretical plates is calculated based on the peak of

icariin as 12143, RSD=0.30%, and tailing factor

T=1.030.

3.2 Solution Preparation

Preparation of reference substance solution:

accurately weigh an appropriate amount of icariin

reference substance, dissolve it with methanol and

dilute to the mark, shake it well, and make a solution

containing about 35μg/ml of icariin.

Preparation of test solution: take about 0.5g of this

product, accurately weigh it, place it in a stoppered

conical flask, accurately add 25ml of dilute ethanol,

close the stopper, weigh it, and ultrasonically treat it

(power 300W, frequency 40kHz) For 30 minutes, it

should let it cool, weigh it again, make up the lost

weight with dilute ethanol, shake well, filter, and take

the additional filtrate to get it.

Preparation of negative control solution: use the

same preparation process to prepare a depleted

negative control sample, and prepare a negative

control solution according to the preparation method

of the test solution.

Preparation of sample recovery test solution: take

0.25g of the test product with the measured content,

accurately weigh it, place it in a stoppered conical

flask, precisely add 15ml of icariin reference

substance solution, and add dilute ethanol 10ml,

dense plug, weigh it, ultrasonic treatment (power

300W, frequency 40kHz) for 30 minutes, let it cool,

then weigh it, use dilute ethanol to make up the lost

weight, shake well, filter, and get it. Six copies should

be prepared in parallel.

Preparation of linear test solution: weigh

accurately 10 mg of icariin reference substance

(content: 94.2%), put it in a 50ml measuring flask,

add methanol to dissolve and dilute to the mark,

shake well, as a reference substance stock solution.

Precisely draw the reference substance stock solution

to prepare a solution containing about 0.20μg,

0.30μg, 0.40μg, 0.50μg, 0.60μg of icariin.

3.3 Determination of the Wavelength is

Detected

By consulting the literature (Li, Chen, Zhang, Wang,

Wang 2020) and the 2020 edition of the Chinese

Pharmacopoeia (National Pharmacopoeia

Commission. 2020) the detection method of icariin in

preparations, the wavelength is 270 nm. Therefore,

the detection method chooses 270nm as the detection

wavelength.

3.4 Methodological Investigation

3.4.1 Specificity

Taking 10μl of icariin reference solution, negative

control solution, and test solution respectively, and it

can be injected into liquid chromatograph for

determination. The chromatograms are shown in

figure 1, figure 2, and figure 3.

It can be seen from the chromatogram that at the

same position as the reference substance retention

time 11.10min, there is no corresponding

chromatographic peak in the negative control

chromatogram. There is a corresponding

chromatographic peak in the chromatogram of the

test product. Therefore, the depleted negative control

and the solvent do not interfere with the

determination.

Figure 1: HPLC chromatogram of icariin reference substance.

Determination of ICARIIN in Traditional Chinese Medicine Preparation by HPLC

873

Figure 2: HPLC chromatogram of negative control.

Figure 3: The HPLC chromatogram of the test sample.

3.4.2 Precision

It can take 10μl of the e solution and inject it into the

high-performance liquid chromatograph, and inject 5

times continuously to measure the peak area. The

results are shown in table 1.

Table 1: Precision test results.

Peak area 1081871 1079740 1079205 1071382 1076269

RSD (%) 0.35

The test results show that the instrument precision

meets the requirements.

3.4.3 Repetitiveness

It should take 10μl of the test solution, inject it into

the high performance liquid chromatograph, repeat

the determination 6 times, and calculate the content

by the external standard method. The results are

shown in table 2.

Table 2: Results of the reproducible findings.

Conten

(%) 0.1847 0.1879 0.1845 0.1852 0.1856 0.1867

Mean conten

(%) 0.1858

RSD (%) 0.70

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

874

The test results show that the reproducibility of

the measurement method meets the requirements.

3.4.4 Sample Recovery Rate

The next step is to take 10μl of the sample recovery

test solution, inject it into the high performance liquid

chromatograph, determine, and calculate the content

by external standard method. The results are shown

in table 3.

Table 3: Results of sample recovery rate.

No.

Sampling

amount

(g)

Content in the

sample

(μg)

Control

addition (μg)

Measurements

(μg)

Rate of

recovery

(%)

Average

recovery rate

(%)

RSD

(%)

1 0.2780 0.2061 0.2125 0.4184 99.91

99.97 0.06

2 0.2762 0.2047

0.2125

0.4170 99.91

3 0.2747 0.2036

0.2125

0.4161 100.00

4 0.2768 0.2052 0.2125 0.4176 99.95

5 0.2727 0.2021 0.2125 0.4147 100.05

6 0.2735 0.2027 0.2125 0.4152 100.00

The test results show that the recovery rate of the

measurement method meets the regulations.

3.4.5 Linear and Range

The next is to take 10 μl each of the linear test

solution and inject it into the high performance liquid

chromatograph, measure the peak area respectively,

and calculate the linear correlation. The results are

shown in table 4.

Table 4: Results of the linear relationship investigation.

Sample size (μg) 0.1888 0.2832 0.3776 0.4720 0.5664

Peak area 455117 697336 930217 1160026 1393169

Taking the injection volume as the abscissa and

the peak area as the ordinate, linear regression is

performed to obtain the regression equation, y =

2.4649×106x -2980.2, and the correlation coefficient

r=0.9999. It can be seen in figure 5.

Figure 4: Linear regression curves.

Determination of ICARIIN in Traditional Chinese Medicine Preparation by HPLC

875

The results show a good linear relationship

between sample size and peak area between 0.1888

μg~0.5664 μg.

3.4.6 Sample Stability

At 0 hour, 2 hours, 4 hours, 8 hours, and 24 hours, 10

μl of the test solution was taken and injected into the

high performance liquid chromatograph to determine

the peak area of icariin. The results are shown in table

Table 5: Results of the stability test.

Time (h) 0 2 4 8 24

Peak area 945015 946455 949716 941619 945068

RSD (%)

0.31

From the test results, the test product solution is

well stable within 24 hours and can meet the

determination needs.

3.5 Sample Determination

According to the determination method and

conditions, three batches of samples were tested, and

the content was calculated by the external standard

method. The results are shown in table 6.

Table 6: Results of epicanosin content determination.

Lot. 20200812 20200813 20200813

Content (%) 0.1850 0.1853 0.1863

Content (mg)

0.7900 0.7916 0.7948

4 DISCUSSION

4.1 In this paper, octadecylsilane-bonded silica gel is

used as the filler, and acetonitrile-water (30:70) is

used as the mobile phase (Wang 2020). The detection

wavelength is 270nm, the flow rate is 1.0ml/min, the

injection volume is 10μl, and the column temperature

is 40°C. The content of icariin in Jiangzhi capsules

was determined by external standard method. The

method’s specificity, accuracy, precision, linearity,

stability all meet the requirements of methodology

verification.

4.2 Solvent (National Pharmacopoeia

Commission. 2020) with dilute ethanol is

environmentally friendly and cheap.

4.3 Icariin was extracted by ultrasound for 20

minutes, 30 minutes, 40 minutes, and 60 minutes and

the measured results remained unchanged, so 30

minutes of ultrasound was used to extract icariin.

4.4 When the sample dosage is 0.2g, 0.5g, 1.0g,

the measured result of icariin remains unchanged, so

0.5g is selected.

4.5 Hyperlipidemia is a disorder of lipid

metabolism and an important risk factor for

atherosclerosis. It can lead to atherosclerotic diseases

such as coronary heart disease, cerebral infarction

and peripheral vascular disease, as well as fatty liver

and acute pancreatitis other diseases. Hyperlipidemia

and atherosclerosis are common in middle-aged and

elderly people. Chinese medicine believes that when

people are over forty years old, the kidney essence

gradually declines, and the qi function is weakened.

The clearing will change from turbidity, and lipid

cohesion will cause hyperlipidemia, qi deficiency and

blood weakness, turbidity, blockage of blood, and

blood stasis atherosclerosis. The disease is located in

the blood vessels, which is a syndrome of deficiency

and deficiency of the underlying condition,

deficiency of the liver, spleen, and kidney are the

roots, the deficiency of the kidney is the mainstay,

and turbid phlegm and blood stasis are the indicators.

Clinical treatment often starts with kidney deficiency

and blood stasis. The treatment is suitable for

warming the kidney and removing turbidity, and

promoting blood circulation to clear the pulse.

Combination of Chinese medicines in Jiangzhi

Capsule, it can warm the kidney and remove turbidity

without injuring the yin, promote blood circulation

and dredge the collaterals without breaking the blood.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

876

5 CONCLUSION

Hyperlipidemia is a disorder of lipid metabolism and

an important risk factor for atherosclerosis. It can lead

to atherosclerotic diseases such as coronary heart

disease, cerebral infarction and peripheral vascular

disease, as well as fatty liver and acute pancreatitis.

The combination of all the medicines in this

prescription can warm the kidney and dissolve

turbidity without injuring the yin. It can also promote

blood circulation and dredge the collaterals without

breaking the blood. As China enters a well-off society

in an all-round way, the improvement of living

conditions and the acceleration of the pace of life

have brought about changes in the dietary structure,

and the population with hyperlipidemia tends to be

younger. In order to facilitate the patient’s use, the

research team explored well-known traditional

Chinese medicine prescriptions. According to the

Chinese Medicine Law of the People’s Republic of

China, the relevant requirements of medical

institutions apply traditional techniques to configure

traditional Chinese medicine preparations. A

controllable technical process route was established

and the internal control agent standard was

formulated with reference to the Chinese

Pharmacopoeia. The internal control standard of this

preparation, through microscopic identification

items, thin-layer identification items, and HPLC

determination of the effective ingredient content of

the main drugs in the preparation, comprehensively

controls the quality of the preparation, and

successfully completes the preparation of the

preparation work. In this study, the traditional

decoction is changed into capsules, the technological

parameters are formulated, and the quality control

indicators are increased. It has made useful

explorations for the protection and discovery of the

proven prescriptions and the promotion of the

modernization of Chinese medicine.

ACKNOWLEDGMENTS

Fund Project: Shandong Provincial TCM Science and

Technology Development Plan Project 2019-0548

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