Chemical constituents comparison of Codonopsis tangshen, Codonopsis
pilosula var. modesta and Codonopsis pilosula
ab
c
Lie-Chwen Lin *, Tung-Hu Tsai and Chao-Lin Kuo
d
a
b
National Research Institute of Chinese Medicine, Taipei, Taiwan, ROC; Graduate Institute of Integrated
Medicine, China Medical University, Taichung, Taiwan, ROC; Institute of Traditional Medicine, National
c
d
Yang-Ming University, Taipei, Taiwan, ROC; School of Chinese Pharmaceutical Sciences and Chinese
Medicine Resources, China Medical University, Taichung, Taiwan, ROC
(Received 16 October 2012; fi nal version received 30 January 2013)
Dang-shen, radix Codonopsis is one of the best-known traditional Chinese
medicines and is used mainly as a tonic agent. Nine commercial products belong
to three species of Codonopsis genus, Codonopsis tangshen, Codonopsis pilosula
var. modesta and a cultivated species of Codonopsis pilosula, which were
purchased from drug stores in Taipei and Taichung, Taiwan. To determine the
chemical differences between samples of Dang-shen, a method combining solvent
partition and HPLC-UV was used. An enriched fraction of n-butanol was obtained,
after partition. Then, the chemical profi le was determined using a C18 reversed
column chromatography in a gradient solvent system with 10–40% acetonitrile in
0.1% formic acid, for 60min. A comparison of the constituents in the nine
commercial products of Dang-shen showed that compounds codonopyrrolidum A, B
and codonoside A only exist in C. tangshen, but not in
C. pilosula var. modesta and C. pilosula. A predominate compound, codonopyrrolidum A,
can be used to distinguish the herbs, C. tangshen from C. pilosula var. modesta and
C. pilosula. Low secondary metabolite content was found in the cultivated species of
C. pilosula.
Keywords: Chemical comparison; HPLC; Campanulaceae; Codonopsis tangshen;
Codonopsis pilosula var. modesta; Codonopsis pilosula; codonopyrrolidum A
1. Introduction
Dang-shen is one of the best-known traditional Chinese medicines and is used frequently as
a tonic agent (Juangsu New Medical College 1979). According to recent pharmacological
research, ‘Dang-shen’ relieves fatigue, increases appetite, enhances immunity, lowers blood
pressure, increases the protection of gastric mucosa cells and improves memory (Wang et al.
1997; Singh et al. 2004). Besides Codonopsis pilosula (Franch.) Nannf. (Campanulaceae), a
series of other Codonopsis species are also used in Chinese medicine, such as C. pilosula
var. modesta (Nannf.) L. T., C. tangshen Oliv., Codonopsis clematidea, Codonopsis moestra,
Codonopsis subglobosa, Codonopsis convolvulacea and Codonopsis mollis (JNMC 1979).
Too many original plants cause complexity in herb regulation. In Taiwan, three types of
Dang-shen are used regularly: C. tangshen, C. pilosula var. modesta and a cultivated
species of C. pilosula (Figure S1). As widely used in the quality control of other herbs,
chromatographic fi ngerprinting is simple and useful. Previous chemical studies of C.
pilosula and C. tangshen have shown the presence of a variety of phenolic glucosides,
neolignan glycoside, alkyl glycosides, diyne and pyrrolidium alkaloids (Mizutani et al. 1988;
Heidrun & Wagner 1994; Tsai & Lin 2008). This
*Corresponding author. Email: lclin@nricm.edu.tw q 2013 Taylor & Francis
2 L.-C. Lin et al.
study used a method combining solvent partition and HPLC-UV to identify the full profi le
fi ngerprint of Dang-shen and to compare the chemical constituents of nine commercial
Dangshen products. Several standard compounds were used in this study. These were
isolated from
C. tangshen. Their structures and spectral data are also reported.
2. Results and discussion
2.1 Identifi cation of isolated compounds
After Dianion HP-20 column and semi-preparative HPLC separation, nine pure compounds,
1–9, were isolated from the EtOH extract of C. tangshen. Compounds 1–9 were identifi ed,
using 1D, 2D NMR and mass data, as codonopyrrolidum B (1), histidine (2), 4-hydroxy
benzoic acid (3), tangshenoside I (4), codonopyrrolidum A (5), lobetyolin (6), codonoside A
(7), tetradeca-4E,8E,12E-triene-10-yne-1,6,7-triol (8) and lobetyol (9) (Figure 1). Then, the
identifi ed compounds 1–9 served as standards in the following analytical experiment.
2.2 Selection of partition systems
The content of secondary metabolite in the herbs is usually low. Liquid–liquid extraction
is the most commonly used method to enhance the relative content of secondary
metabolites in the crude extract. From a previous study (Hu et al. 2002), it was found
that C. tangshen contains a lot of carbohydrate, which causes dilution and reduces the
relative response of other chemical constituents, during analysis. Therefore, removing the
majority carbohydrate to increase the relative content of other constituents was very
important in the following analysis. Four partition systems of n-BuOH/H2O, sat. n-BuOH
(n-BuOH pre-saturated with H2O)/H2O, CH3Cl/H2O and sat. CH3Cl (CH3Cl pre-saturated with
H2O)/H2O were tested. Figure S2 shows the chromatograms of each partition layer, which
were obtained using a C18 reversed column chromatography in a gradient solvent system
of 10–40% acetonitrile in 0.1% formic acid, over
O
HO
HO OH OH H3CO H OH3CO
OH
OH O
OH O
O HO O
O HO+ HO
OH HO+ N
O
OHH N1 H3CO OH OMe OMe
1: Codonopyrrolidium B 4: Tangshenoside I 5: Codonopyrrolidium A
H
H3C
OH O
HH
H HO
HO
OH
OH
HO
HO
O
OH
HH
O O OH1"
H3CO
H3CO
O
O
H3C
O
OO
HO
OH
OH O OH
OHH
6'''
OH
OH
6: Lobetyolin
7: Codonoside A
H
9: Lobetyol
OH
8: Tetradeca-4E,8E,12E-triene-10-yne-1,6,7-triol
Figure 1. Chemical structures of compounds 1, 4–9.
Natural Product Research
60 min. Careful examination of the chromatograms reveals that the sat. n-BuOH layer
(Figure S2D) presents a much better peak shape and intensity than the n-BuOH layer
(Figure S2B). Finally, the partition system, sat. n-BuOH/H2O, was selected and the
chemical profi les of Dangshen herbs were traced on their sat. n-BuOH extract.
2.3 HPLC analysis of Dang-shen
The chemical constituents of extracts in the chromatograms were characterized by
comparison with the retention time and UV spectrum of standard compounds. The retention
time of standards 1–9 was about 4.87 (1), 7.12 (2), 14.48 (3), 18.65 (4), 20.24 (5), 32.95
(6), 33.76 (7),
41.64 (8) and 42.65 min (9). Figure S3 shows a comparison of the profi les of sat.
n-BuOH extract obtained from nine Dang-shen products. The compounds lobetyolin (6),
tetradeca-4E,8E,12Etriene-10-yne-1,6,7-triol (8) and lobetyol (9) appeared in both species of
C. tangshen (Figures S3A, S3D and S3G) and C. pilosula var. modesta (Figures S3B, S3E
and S3H), but rare or not at all in the cultivated species of C. pilosula (Figures S3C, S3F
and S3I). In the China Pharmacopoeia 2010 edition (China Pharmacopoeia 2010),
lobetyolin is used as a standard compound to monitor the quality of Dang-shen material.
From this view point, both C. tangshen and C. pilosula var. modesta seem to achieve the
required standard, while their chemical pro fi les are very different. The compounds
codonopyrrolidum B (1), codonopyrrolidum A (5) and codonoside A (7) only appear in C.
tangshen (Figures S3A, S3D and S3G). These compounds, especially the abundant
codonopyrrolidum A (5), can be used as differentiation markers between
C. tangshen, C. pilosula var. modesta and a cultivated species of C. pilosula. A reduced
content was observed in the chromatogram of the cultivated species of C. pilosula. The
cultivation time of the cultivated species of C. pilosula is relatively shorter than the other
two. This may be a reason to cause low content of secondary metabolite in the cultivated
species of C. pilosula,oran arti fi cial cultivation environment may have affected the
production of secondary metabolites. The results show that the quality of the cultivated
species of C. pilosula is the worst of all nine Dang-shen products.
3. Conclusions
A method combining liquid–liquid partition treatment and HPLC analysis was used, which
clearly distinguishes different species of Dang-shen. Nine chemical constituents were identifi
ed from C. tangshen, of which the compound codonopyrrolidum A (5) can be used as a
marker to differentiate C. tangshen, C. pilosula var. modesta and a cultivated species of C.
pilosula.
Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1–S3.
Acknowledgements
Funding for this study was provided in part by research grants from the National Research Institute of Chinese
Medicine; and the National Science Council, Republic of China (NSC 100-2320-B-077-002MY2).
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