simultaneous chemical fingerprinting and quantitative analysis of crude and processed radix...

Research Article

Simultaneous chemical fingerprinting andquantitative analysis of crude and processedRadix Scrophulariae from different locationsin China by HPLC

A validated liquid chromatography method was first developed to evaluate the quality of

crude and processed Radix Scrophulariae extracts through establishing chromatographic

fingerprint and simultaneous determination of five bioactive compounds, namely

5-hydroxymethylfurfural (5-HMF), acteoside, angroside C, harpagoside and cinnamic

acid. The chromatographic were separated on an Agilent Zorbax Extend C18 column

(250 mm� 4.6 mm, 5 mm) and detected by diode array detector (DAD). Mobile phase was

composed of (A) aqueous phosphoric acid (0.03%, v/v) and (B) acetonitrile using a

gradient elution. Analytes were performed at 301C with a flow rate of 1.0 mL/min and UV

detection at 280 nm. All calibration curves showed good linear regression (r2Z0.9996)

within the tested ranges, and the recovery of the method was in the range of

98.12–103.38%, with RSD values ranging from 0.6 to 2.8%. In addition, the contents of

those five bioactive compounds in crude and processed Radix Scrophulariae prepared by

different locations of China were determined to establish the effectiveness of the method.

The results demonstrate that the developed method is accurate and reproducible and

could be readily utilized as a suitable quality control method for the quantification of

Radix Scrophulariae.

Keywords: Chemical fingerprinting / HPLC / Processed / Radix ScrophulariaeDOI 10.1002/jssc.201100136

1 Introduction

Radix Scrophulariae (Xuanshen in Chinese), derived from

the dried root of Scrophularia ningpoensis Hemsl., is one of the

oldest and most frequently used Chinese herbs for oriental

medicine in China. The crude drug and its processed

products of zheng zhi pin (ZZP) are used clinically [1–4].

Pharmacological studies and clinical practice have demon-

strated that Radix Scrophulariae possesses various bioactiv-

ities, including anti-chronic inflammatory, antihypertensive,

abirritative, antispasmodic, anti-HBV and immunological

enhancement. Harpagoside is a characteristic and active

constituent of Radix Scrophulariae and some researches also

showed that harpagoside has some pharmacological effects

which are closely related to nourishment of yin for lowering

fire [5–6]. In China, Radix Scrophulariae is mainly distributed

in Zhejiang, Sichuan, Hebei, Jiangsu, Fujian, Gansu and so

on. It is, therefore, desirable to determine a reliable and

accurate methodology to differentiate the samples collected

from these areas. Although some studies on the fingerprints

of Radix Scrophulariae using HPLC have been published,

none of them compared the processed product of Radix

Scrophulariae from different areas in China [7–8]. Chromato-

graphic fingerprint analysis by which multiple compounds in

single herbal drugs and finished traditional Chinese medi-

cine (TCM) can be identified represents a rational approach

for the quality assessment of TCM. It utilizes chromato-

graphic techniques, such as UPLC, GC, HPLC, HPTLC, etc.

[9], to construct specific patterns of recognition for multiple

compounds in TCM. The entire pattern of compounds can

then be evaluated to determine not only the absence or

presence of desired markers or activities but the complete set

of ratios of all detectable analytes [10–11]. Thus, chromato-

graphic fingerprint analysis of herbal drugs represents a

comprehensive qualitative approach for the purpose

of species authentication, evaluation of quality, and ensuring

the consistency and stability of TCM and their related

products. There were lots of literature about the quality

control of Radix Scrophulariae and its preparations, but

Yun Zhang1�

Gang Cao�

Jinyu Ji1

Xiaodong Cong1

Shengbo Wang1

Baochang Cai1,2

1Research Center of TCMProcessing Technology,Zhejiang Chinese MedicalUniversity, Hangzhou,P. R. China

2Engineering Center of StateMinistry of Education forStandardization of ChineseMedicine Processing, NanjingUniversity of Chinese Medicine,Nanjing, P. R. China

Received February 19, 2011Revised March 10, 2011Accepted March 16, 2011

Abbreviations: 5-HMF, 5-hydroxymethylfurfural; RPA,relative peak area; RRT, relative retention time; TCM,traditional Chinese medicine; ZZP, zheng zhi pin �These authors contributed equally to the work.

Correspondence: Prof. Baochang Cai, Engineering Center ofState Ministry of Education for Standardization of ChineseMedical Processing, Nanjing University of Chinese Medicine,Nanjing 210029, P. R. ChinaE-mail: [email protected]: 186-25-8679-8281

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2011, 34, 1429–1436 1429

literature about combination of chromatographic fingerprint

and quantification of multi-ingredients for the quality control

of processed Radix Scrophulariae has not been published.

The present study describes a simple, accurate and

practical HPLC method for chromatographic fingerprint

analysis and simultaneous quantification of five compounds

(5-hydroxymethylfurfural (5-HMF), acteoside, angroside C,

harpagoside and cinnamic acid) in crude and processed

Radix Scrophulariae. Chromatographic conditions and

method validation were objectives of the research reported.

The method was successfully used for evaluating the quality

of Radix Scrophulariae and its processed product grown in

China. In addition, the identification of marker compounds

for classification and evaluation of the quality of crude and

processed extracts were performed by means of the RP-

HPLC analysis.

2 Materials and methods

2.1 Samples, chemicals and reagents

A total of 44 crude Radix Scrophulariae from different areas

in China were investigated and collected (Table 1). These

herbal samples were authenticated by Professor Yun Zhang

(Research Center of TCM Processing Technology, Zhejiang

Chinese Medical University). Voucher specimens were

stored at the Research Center of TCM Processing Technol-

ogy. The reference standards 5-HMF, acteoside, angroside

C, harpagoside and cinnamic acid were purchased from the

National Institute for the Control of Pharmaceutical and

Biological Products. The chemical structures of these five

compounds (Fig. 1) were confirmed by 1H, 13C nuclear

magnetic resonance (NMR) spectroscopy and mass spec-

trum, and purities were over 98% by HPLC analysis.

The HPLC grade acetonitrile was purchased from

Merck (Darmstadt, Germany), phosphoric acid was

purchased from Tedia (OH, USA), and ultrapure water was

prepared by a Milli-Q50 SP Reagent Water System (Milli-

pore, MA, USA) for the preparation of samples and buffer

solutions. Other reagents were of analytical grade.

2.2 Instrumentation and HPLC conditions

Analyses were performed on Agilent Series 1200 liquid

chromatograph (Agilent Technologies, Palo Alto, CA, USA)

with diode array detector. Detection wavelengths were set

at 280 nm. An Agilent Zorbax Extend C18 (250 mm� 4.6

mm, 5 mm) was used with a flow rate of 1.0 mL/min. The

injection volume was 10 mL and the column temperature

was maintained at 301C; mobile phase was composed of (A)

aqueous phosphoric acid (0.03%, v/v) and (B) acetonitrile

using a gradient elution of 3–6% B at 0–8 min, 6–15% B at

8–18 min, 15–20% B at 18–25 min, 20–35% B at 25–35 min,

35–47% B at 35–38 min, 47% B at 38–40 min, 47–75% B at

40–45 min and 75–80% B at 45–50 min.

2.3 Standard solution preparation

Primary stock standard solutions of the five compounds were

prepared by dissolving them with methanol, respectively, to

get a concentration of 0.034 mg/mL (5-HMF), 0.0543 mg/mL

(acteoside), 0.0284 mg/mL (angroside C), 0.0204 mg/mL

(harpagoside) and 0.0275 mg/mL (cinnamic acid). Working

mixed standard solutions were prepared daily by mixing and

Table 1. Crude and processed materials used in this work and

the similarity values of 44 chromatograms of Radix

Scrophulariae

Sample

no.

Origin Similarities�

(crude samples)

Similarities�

(processed

samples)

1 Dongyang, Zhejiang A 1.000 1.000

2 Dongyang, Zhejiang B 0.943 0.972

3 Dongyang, Zhejiang C 0.987 0.975

4 Dongyang, Zhejiang D 0.97 0.964

5 Dongyang, Zhejiang E 0.895 0.972

6 Dongyang, Zhejiang F 0.955 0.952

7 Panan, Zhejiang A 0.973 0.976

8 Henan 0.927 0.988

9 Dongyang, Zhejiang G 0.987 0.996

10 Hunan 0.985 0.972

11 Dongyang, Zhejiang H 0.981 0.958

12 Anhui 0.859 0.984

13 Panan, Zhejiang B 0.961 0.978

14 Dongyang, Zhejiang I 0.948 0.987

15 Panan, Zhejiang C 0.961 0.965

16 Dongyang, Zhejiang J 0.963 0.981

17 Xianju, Zhejiang A 0.985 0.979

18 Xianju, Zhejiang B 0.967 0.924

19 Chongqing 0.962 0.983

20 Shandong A 0.945 0.973

21 Dongyang, Zhejiang K 0.96 0.977

22 Jinyun, Zhejiang A 0.973 0.97

23 Jinyun, Zhejiang B 0.972 0.973

24 Jinyun, Zhejiang C 0.94 0.969

25 Jinyun, Zhejiang D 0.903 0.971

26 Jinyun, Zhejiang E 0.988 0.987

27 Jinyun, Zhejiang F 0.979 0.984

28 Panan, Zhejiang D 0.956 0.976

29 Panan, Zhejiang E 0.958 0.966

30 Panan, Zhejiang F 0.948 0.978

31 Panan, Zhejiang G 0.961 0.977

32 Fuyang, Zhejiang 0.973 0.974

33 Zhejiang I 0.945 0.972

34 Henan 0.959 0.975

35 Shandong B 0.939 0.966

36 Zhejiang II 0.947 0.98

37 Jiangxi 0.965 0.977

38 Hubei 0.933 0.992

39 Zhejiang III 0.959 0.971

40 Zhejiang IV 0.867 0.972

41 Zhejiang V 0.984 0.966

42 Shangdong C 0.981 0.997

43 Shangdong D 0.97 0.965

44 Zhejiang VI 0.961 0.98

J. Sep. Sci. 2011, 34, 1429–14361430 Y. Zhang et al.


diluting the stock solutions with methanol. The standard

stock and working solutions were all prepared in calibrated

flasks and stored at 41C. All calibration curves were

constructed from peak areas of the reference standards versus

their concentrations. The solutions were filtered through a

0.45-mm membrane prior to injection. The RP-HPLC

chromatograms of mixed standards is shown in Fig. 2.

2.4 Preparation of sample solutions

The powder of crude and processed Radix Scrophulariae

samples were precisely weighed (0.500 g), and transferred

into dark brown calibrated flasks. They were extracted with

50 mL of 50% methanol in an ultrasonic bath for 45 min.

Additional 50% methanol was added to make up the lost.

The supernatants were filtered through a 0.45-mm

membrane prior to injection.

2.5 Data analysis and acquisition of characteristic

information

The similarity evaluation system for chromatographic finger-

print of TCM was developed by the Chinese Pharmacopoeia

Commission in 2004 in Beijing; it can reflect the similarity of

the distribution ratio of the chemical composition accurately,

rather than a function of the quantitative evaluation. The

professional software was used to evaluate the similarity of

TCM fingerprint profiles in specific accordance with the state

regulations of China and was purchased from the Chinese

Pharmacopoeia Commission.

3 Results and discussion

3.1 Optimization of chromatographic conditions

In order to obtain better chromatograms, different mobile phase

compositions, including water–acetonitrile, water–methanol,

aqueous phosphoric acid (0.01%, v/v)–acetonitrile, aqueous

phosphoric acid (0.03%, v/v)–acetonitrile were tested. As a

result, the combination of aqueous phosphoric acid (0.03%,

v/v)–acetonitrile for mobile phase was the best for separation.

Furthermore, other chromatographic variables were also

optimized, including analytical columns (Hanbon Lichrospher

C18 and Agilent Zorbax Extend C18), the column temperatures

(20, 25 and 301C) and the flow rates (0.5, 0.8 and 1.0 mL/min).

Eventually, the optimal separation was achieved on an Agilent

OH

O

OOH

O

H

C

O

O

HO OH

Me

O

OO OH

OHHO

OH

cinnamic acid 5-HMF harpagoside

OMe

HO

HOOH

OO

O

O

OH

O

OMe

OH

O

HO

O

Me O

HO

HO

OH

OMe

HO

HOOH

OO

OOH

OH

O

OH

OHC

O

HO

HO

angroside C acteoside

Figure 1. The chemical structures of five active components in Radix Scrophulariae.

J. Sep. Sci. 2011, 34, 1429–1436 Liquid Chromatography 1431


Zorbax Extend C18 column (250 mm� 4.6 mm, 5 mm) at a

column temperature of 301C with a flow rate of 1.0 mL/min.

To obtain optimal extraction efficiency, a previous study

had chosen ethanol as the extraction solvent. Simulta-

neously, in the present study, extraction methods (ultrasonic

water bath), extraction time (45 min), temperature (251C)

and concentration of ethanol aqueous solution (50%, v/v)

had also been selected. These findings are in accordance

with the results previously reported.

3.2 Analytical method validation

The HPLC method was validated by defining the linearity,

limits of quantification and detection, identification and

quantification of the analytes, repeatability, precision,

stability and recovery.

Calibration working standard solutions were freshly

prepared in methanol by appropriate dilution of the stock

solutions. All calibration curves were constructed from peak

areas of the reference standards versus their concentrations.

The peak area values were the average values of three

replicate injections. The results of calibration are summar-

ized in Table 2, and a good correlation was found between

the peak area (y) and the concentrations (x) (r2Z0.9996) for

all the compounds in the range of concentration tested at

their detected wavelengths.

Injection precision was assessed by repetitive injection

of the same sample solution six times in a day. The results

are shown in Table 3. From Table 3, it appears that the RSD

of RRT and RPA did not exceed 0.33 and 0.18%, respec-

tively.

To further evaluate the repeatability of the developed

assay, crude and processed Radix Scrophulariae were

analyzed in six replicates as described above. The RSDs wereFigure 2. HPLC chromatograms of mixed standards.

Table 2. Calibration plots, LOQ and LOD for the five analyses

Marker compound Regression equation r2 Linear range (mg/mL) LOQ (mg/mL) LOD (mg/mL)

Acteoside y 5 2233.28x�0.7159 0.9999 6.78–81.45 0.58 0.16

Angroside C y 5 4126.60x�04145 0.9998 3.55–42.60 0.26 0.07

Harpagoside y 5 24831.93x�0.071 0.9999 2.55–30.60 0.14 0.05

Cinnamic acid y 5 17970.24x�1.2931 0.9999 3.44–41.25 0.35 0.09

5-HMF y 5 16688.64x10.3185 0.9996 4.25–51.00 0.33 0.11

Table 3. Results of relative retention times and relative peak areas of precision test, repeatability test and stability test on LC fingerprint

of Radix Scrophulariae populations from different locations in China

Peak no. Relative retention time (RRT) Relative peak area (RPA)

Mean (relative standard deviation (RSD) %) Mean (RSD%)

Rpt Rrt Rst Rcp Rpt Rrt Rst Rcp

1 0.07 (0.01) 0.07 (0.00) 0.07 (0.01) 0.07 (0.00) 0.32 (0.02) 0.40 (0.14) 0.32 (0.01) 0.34 (0.47)

2 0.08 (0.01) 0.08 (0.01) 0.08 (0.01) 0.09 (0.00) 3.34 (0.03) 0.62 (0.17) 3.33 (0.03) 2.15 (0.31)

3 0.11 (0.07) 0.11 (0.03) 0.11 (0.01) 0.11 (0.00) 0.31(0.18) 0.45 (0.43) 0.34 (0.16) 0.18 (0.32)

4 0.92 (0.10) 0.92 (0.00) 0.92 (0.00) 0.92 (0.00) 0.45 (0.08) 0.44 (0.10) 0.44 (0.08) 0.62 (0.46)

5 0.96 (0.14) 0.96 (0.00) 0.96 (0.00) 0.93 (0.01) 0.33 (0.07) 0.32 (0.04) 0.32 (0.04) 0.11 (0.25)

6 1.00 (0.33) 1.00 (0.00) 1.00 (0.00) 1.00 (0.00) 1.00 (0.00) 1.00 (0.00) 1.00 (0.00) 1.00 (0.00)

7 1.13 (0.09) 1.13 (0.00) 1.13 (0.00) 1.12 (0.00) 2.66 (0.01) 2.64 (0.01) 2.68 (0.01) 3.31 (0.29)

8 1.21 (0.12) 1.21 (0.03) 1.22 (0.00) 1.23 (0.00) 0.21 (0.05) 3.27 (0.01) 3.31 (0.01) 2.89 (0.23)

9 1.40 (0.21) 1.39 (0.14) 1.39 (0.00) 1.41 (0.00) 0.18 (0.07) 0.16 (0.05) 0.18 (0.06) 0.14 (0.38)

10 1.40 (0.16) 1.40 (0.24) 0.07 (0.00) 1.42 (0.00) 0.41 (0.01) 0.38 (0.03) 0.41 (0.02) 0.42 (0.37)

11 1.41 (0.32) 1.41 (0.47) 0.08 (0.00) 1.42 (0.00) 0.13 (0.02) 0.12 (0.02) 0.13 (0.03) 0.18 (0.37)

Rpt (n 5 6), Rrt (n 5 6) and Rst (n 5 6): determine the same sample solution at 0, 2, 4, 6, 8, 12, 24 and 48 h after it was prepared,

respectively) and Rcp represent the results of relative retention times and relative peak areas of precision test, repeatability test and

stability test on LC fingerprint of Radix Scrophulariae populations from different locations in China, respectively



taken as measurements of repeatability. The results are

shown in Table 3. From Table 3, the RSDs of RRT and RPA

were not more than 0.47 and 0.43% for all analytes,

respectively.

Stability was tested with crude and processed Radix

Scrophulariae at room temperature and analyzed at 0, 2, 4,

8, 12, 24 and 48 h within 2 days, respectively. The results are

shown in Table 3. The RSD of RRT and RPA were less than

0.01 and 0.16% for all analytes, respectively. The similarity

of these results indicated that the sample remained stable

during this period.

Accuracy was determined by the recovery test. An

appropriate amount of Radix Scrophulariae powder was

weighed and spiked with a known amount of each standard

compound. They were then treated and analyzed as descri-

bed above. Each sample was analyzed in six replicates. The

total amount of each analyte was calculated from the

corresponding calibration curve. Mean recoveries of five

compounds were 98.12–103.38%, with RSD values ranging

from 0.6 to 2.8% (n 5 6).

3.3 Analysis of chromatogram of crude and

processed Radix Scrophulariae

The chromatographic fingerprints showed abundant diver-

sity of chemical constituents that were analyzed by

authentication and quantification in crude and processed

Radix Scrophulariae from different populations. In this

paper, an LC chemical fingerprinting method was

utilized and developed for quality control of crude and

processed Radix Scrophulariae. The fingerprinting analysis

was operated through a software Similarity Evaluation

System for Chromatographic Fingerprint of TCM (Version

2004A), which was recommended by SFDA. To perform it,

the chromatograms of different samples have to be

standardized. The process of standardization included

the selection of ‘‘common peaks’’ in chromatograms and

the normalization of retention times of all the common

peaks. Furthermore, the total area of the common peaks

must be more than 90% of the whole area in one

chromatogram. Here, the extracts of 44 batches of Radix

Figure 3. The comparison ofchromatographic fingerprintsof crude (A) and processed (B)Radix Scrophulariae collectedfrom 44 origins.



Scrophulariae samples collected from different locations

served as the sample set. The LC fingerprints are shown

in Fig. 3.

A total of 11 and 14 common peaks were marked as the

common peaks in the chromatograms in a total of 44 crude

and processed Radix Scrophulariae, respectively (Fig. 4).

The RRT and relative retention area (RPA) of these 11 and

14 common peaks with respect to peak 6 and peak 9 in 44

crude and processed samples are shown in Table 3,

respectively.

Five peaks were identified using standards. In the

present study, according to the contents and pharmacological

properties of major constituents in crude and its processed

product, the peaks of number 4, 6, 7 and 8 in crude drug are

acteoside (29.681 min), angroside C (32.188 min), harpago-

side A (36.091 min) and cinnamic acid (39.462 min), respec-

tively. Simultaneously, the peaks of number 5, 7, 9, 10 and 11

in processed drug are 5-HMF (10.848 min), acteoside

(29.660 min), angroside C (32.196 min), harpagoside A

(36.115 min) and cinnamic acid (39.431 min), respectively.

Altogether, 44 samples of crude or processed Radix

Scrophulariae were analyzed with the procedure developed,

respectively. These samples were collected from a variety of

geographical locations. Similarities among the 44 samples

of crude or processed Radix were calculated using the

similarity evaluation system recommended by SFDA. The

results indicated that their chromatographic patterns were

generally consistent, although the absorption intensity of

some peaks was different. The parameters evaluated and

validation aspects are different from the general assaying

methods. Authentication and identification of a herbal

medicine can be accurately carried out using chromato-

graphic fingerprints, even if batches or concentrations are

not the same in different samples.

3.4 Simultaneous quantification of five compounds

in crude and ZZP samples

The developed analytical method was successfully applied to

the simultaneous determination of 5-HMF, acteoside,

angroside C, harpagoside and cinnamic acid in 44 samples

of crude and ZZP samples, which were obtained from

various provinces and cities in China, respectively.

The contents of above five compounds in crude and

processed Radix Scrophulariae samples analyzed are listed

4: acteoside

6: angroside C

7: harpagoside

8: cinnamic acid

5: 5-HMF

7: acteoside

9: angroside C

10: harpagoside

11: cinnamic acid

A

B

Figure 4. Characteristic peaks in the LCchromatograms of the extracts of crude (A)and processed (B) Radix Scrophulariaecollected from 44 origins.



in Table 4. The results showed that the content of each

compound in crude and processed drugs varied signifi-

cantly. For instance, for Radix Scrophulariae collected from

Hunan, the content of harpagside A was lower in crude

drug (1.26 mg/g), but higher in processed drug (3.15 mg/g),

5-HMF was hardly detected in crude drug, but

higher in processed Radix Scrophulariae (1.54 mg/g), the

contents of acteoside, angroside C and cinnamic acid in

crude drug (1.24, 2.23 and 1.21 mg/g) were higher

than those in processed samples (0.079, 1.54 and 0.99

mg/g). The variations might result from different

processing procedures for Radix Scrophulariae. It was

reported that processing or heating could drastically

increase the content of 5-HMF. It could also be seen that the

total contents of five compounds varied slightly in the

same type of samples from different suppliers, which might

be due to the differences in soils and climates in each

region. Thus, it is necessary to control the main active

Table 4. Amounts of the five compounds in crude and processed Radix Scrophulariae populations from different locations in China

Sample Amount (mg/g) – Crude sample Amount (mg/g) – ZZP sample

Acteoside Angroside C Harpagoside Cinnamic acid Acteoside Angroside C Harpagoside Cinnamic acid 5-HMF

1 1.54 2.70 1.30 1.12 2.47 3.73 1.15 1.72 1.06

2 4.87 3.78 1.80 2.60 3.06 4.28 1.87 2.92 0.99

3 0.98 2.06 1.74 1.76 1.21 3.11 1.68 2.54 0.77

4 2.86 4.11 1.66 2.21 2.80 3.45 1.42 3.05 1.44

5 3.64 5.29 2.21 0.95 2.33 4.40 1.06 1.5395 0.85

6 3.54 4.15 2.90 2.69 2.96 4.18 1.39 2.96 2.77

7 3.33 4.10 1.55 1.77 1.96 4.04 1.62 3.11 1.32

8 2.09 2.65 1.02 2.06 1.35 2.73 0.73 2.25 2.26

9 2.11 3.35 1.85 1.58 2.66 3.65 1.26 2.23 1.20

10 1.24 2.230 1.26 1.21 1.54 3.15 0.99 1.54 0.79

11 2.13 2.90 2.18 2.19 1.28 3.00 1.66 2.52 0.77

12 4.91 2.82 0.71 2.46 3.35 3.97 1.14 3.03 1.18

13 7.14 3.46 1.70 1.81 4.40 4.32 1.41 2.35 1.63

14 2.42 4.06 1.94 3.11 2.42 4.06 1.57 3.06 1.57

15 5.77 3.75 2.00 2.45 3.82 4.38 1.62 2.69 1.35

16 4.48 3.74 1.32 1.86 1.98 3.74 1.39 3.07 0.91

17 1.50 2.21 1.36 1.35 2.25 3.47 1.18 2.21 1.72

18 4.20 3.13 1.42 2.02 3.70 4.01 1.27 2.41 1.81

19 2.78 2.93 1.41 2.23 1.27 2.69 0.73 2.27 2.25

20 5.32 3.30 1.49 2.30 3.79 4.19 1.49 2.64 1.59

21 5.54 3.56 1.69 2.40 2.70 4.11 1.49 2.97 2.14

22 5.35 3.74 2.08 2.16 3.29 9.57 1.01 1.90 0.18

23 5.21 3.97 2.22 2.13 2.87 4.18 1.77 3.01 1.27

24 5.49 2.88 1.27 2.12 2.64 3.66 1.53 3.15 1.48

25 5.41 2.93 1.11 2.52 2.80 3.85 1.34 2.99 1.33

26 1.67 2.71 1.57 1.29 1.65 3.59 1.378 2.39 0.89

27 1.46 2.39 1.02 1.92 1.01 2.88 1.00 2.38 1.62

28 1.66 3.14 2.02 1.95 1.10 3.09 1.58 2.65 0.73

29 4.56 4.45 3.01 2.69 3.60 4.06 1.42 2.69 2.16

30 7.43 3.69 1.75 2.10 4.21 4.59 1.56 2.63 1.56

31 5.56 3.60 1.80 2.53 3.62 4.20 1.63 2.72 1.32

32 6.00 3.75 1.49 2.01 3.33 3.74 1.40 2.75 1.49

33 3.22 4.14 2.03 2.40 2.70 3.63 1.40 3.03 2.06

34 4.89 4.03 2.17 2.78 3.16 4.21 1.64 2.98 1.98

35 3.92 3.10 2.37 2.54 3.39 4.18 1.49 2.80 2.06

36 4.53 3.33 2.45 2.77 5.00 6.27 2.59 4.13 1.75

37 4.60 2.81 2.33 2.48 3.37 3.97 1.54 2.94 1.36

38 2.86 3.25 2.29 2.49 2.95 3.55 1.60 2.96 1.16

39 8.63 3.22 1.39 2.10 3.83 3.97 1.26 2.66 1.37

40 4.38 6.11 0.51 1.488 3.15 3.99 1.29 2.99 2.13

41 4.96 2.61 0.77 2.41 3.73 4.34 1.44 2.86 1.25

42 1.50 2.19 1.59 1.87 2.11 3.20 0.93 1.89 1.55

43 1.20 1.93 1.90 1.62 2.06 3.33 1.32 1.94 0.92

44 1.35 1.86 1.73 1.93 1.35 2.72 1.08 2.34 1.62



components in Radix Scrophulariae by good agricultural

practice (GAP) and the norm of Chinese medicinal mate-

rials processing.

4 Concluding remarks

In the present study, a simple, accurate and reliable method

was developed to evaluate the quality of crude and processed

Radix Scrophulariae through establishing chromatographic

fingerprint and simultaneous determination of five bioactive

compounds, namely acteoside, angroside C, harpagoside,

cinnamic acid and 5-HMF. The results demonstrate that the

developed method is accurate and reproducible and could be

readily utilized as a suitable quality control method for the

quantification of Radix Scrophulariae. These findings and

results also provide a strong basis to establish good

agriculture practice and select geo-authentic crude drug

for Radix Scrophulariae.

The authors are grateful for the financial support of themedical science research fund of the Minister of Health of thePeople’s Republic of China (WKJ2010-2-019).

The authors have declared no conflict of interest.

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