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J Acupunct Res > Volume 32(2); 2015 > Article
Lee, Kim, Lee, Kim, Lee, and Kim: A Study on Standardization of Shinbaro Pharmacopuncture Using Herbal Medicines Identification Test and HPLC-DAD
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The present study was an evaluation and standardization of herbal components in order to establish the efficacy and safety of Shinbaro pharmacopuncture.


Among the raw materials of Shinbaro pharmacopuncture, the components Cibotii Rhizoma, Eucommiae Cortex, and Ledebouriellae Radix were assessed through ingredient verification experiments using thin-layer chromatography(TLC) and ultraviolet rays(UV) lamps. In addition, we standardized Acanthopanacis Cortex and Achyranthis Radix through validation using high performance liquid chromatograph-diode array detector(HPLC-DAD).


As result appeared a blue-white fluorescence under ultraviolet rays; changed to dark green after adding 1 % ferric chloride solution(due to Cibotii Rhizoma), and presented a yellow-green fluorescence when mixed with an ethyl ether under UV lamps by way of the ethyl ether layer, confirming Eucommiae Cortex. Ledebouriellae Radix was confirmed as dark brown spots at Rf values of 0.56 and 0.71 using TLC. Additionally, Acanthopanacis Cortex and Achyranthis Radix HPLC test results showed that linearity was R2≥0.99, and detection limit and quantitation limit were 0.23 to 1.29 μg/mL, and 0.71 to 3.90 μg/mL, respectively. Furthermore, precision and accuracy were confirmed to have relative standard deviation(RSD) values of 0.10 to 1.89 % and 96.19 to 103.72 %, respectively. Shinbaro pharmacopuncture did not have any overlapping or interference from other peaks in detection under the above-mentioned analysis conditions.


In conclusion, we confirmed that maintenance of Shinbaro pharmacopuncture validity was possible by means of quality control of Cibotii Rhizoma, Eucommiae Cortex, and Ledebouriellae Radix through ingredient identification and Acanthopanacis Cortex and Achyranthis Radix through high performance liquid chromatograph(HPLC) analysis. Further, we hope to contribute to the development strategy of herbal industry acupuncture.

Fig. 1.
Identification test of herbal medicine
Fig. 2.
TLC chromatogram of ➀ Shinbaro pharmacopuncture and ➁ references of Eucommiae Cortex
Eluent system :Cloroform: Methanol Water(v/v).
Fig. 3.
HPLC chromatogram by preparation method
a: acanthoside D at standard(205 nm),
b: 20-hydroxyecdysone at standard(246 nm),
c: acanthoside D at Shinbaro pharmacopuncture(205nm),
d: 20-hydroxyecdysone at Shinbaro pharmacopuncture(246 nm).
Table 1.
The Linearity, Regression Equation, Correlation Coefficient(R2), and LOD and LOQ for Measurement of Standard Components by HPLC
Components Regression equation R2 (n=3) LOD (μg/mL) LOQ (μg/mL)
Acanthoside D Y=111253x + 54117 0.9998 0.30 0.90
20-hydroxcy ecdysone Y=42691x − 6580 0.9994 0.23 0.71

Y: peak area. x: amount(μg/mL).

LOD: limit of detection. 3.3×(SD of the response/slope of the calibration curve.

LOQ: limit of quantitation. 10×(SD of the response/slope of the calibration curve.

Table 2.
Within-run and between-run Variations of Standard Materials by HPLC-DAD
Standard materials Retention time(min) Concentration (μg/mL) Within-run(n=4) Between-run(n=3)
Mean±SD (μg/mL) RSD(%) Mean±SD (μg/mL) RSD(%)
Acanthoside D 108.62 5 5.16 ± 0.22 1.08 5.21 ± 0.02 0.10
10 9.82 ± 0.52 1.32 9.77 ± 0.14 0.46
50 50.02 ± 1.81 0.90 50.03 ± 0.52 0.35
20-hydroxcye cdysone 22.63 5 5.20 ± 0.19 0.89 5.14 ± 0.22 1.42
10 9.54 ± 0.30 0.79 9.65 ± 0.08 0.26
50 50.05 ± 1.72 0.86 50.04 ± 0.38 0.25


Table 3.
Analytical Results of Accuracy Test for Standard Materials by HPLC
Components Spiked amount (μg/mL) Measured amount (μg/mL, n=4) RSD (%) Recovery (%)
Acanthoside D 5 5.19 1.02 103.72
10 9.79 1.86 97.90
50 50.02 0.50 100.05
20-hydrox yecdysone 5 5.03 0.87 100.55
10 9.62 0.97 96.19
50 50.04 0.92 100.08

Recovery(%)=[(amount found-original amount)/amount spiked] ×100 %.



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