Metabonomic approach to Molecular Mechanism of Detoxic
Eﬀect of Compatibility of Traditional Chinese Medicine Part I: Toxic Components of Euphorbia kansui
Name of dispatched researcher（派遣研究者名）

Lei Zhang

Instructor（指導教官，研究指導者）

Jian-xin LiAfÀliation of Instructor（所属）

School of Chemistry and Chemical
Engineering, Nanjing University

Host Collaborator（受入研究者）

Shigetoshi Kadota

Abstract:
The roots of Euphorbia kansui (EK) and the fruits of Zizyphus jujuba (ZJ) have beenclinically used in traditional Chinese medicine (TCM). According to the TCM theory, EK has
been frequently used together with ZJ as a drug-pair (named as compatibility), or processed withvinegar (VEK) in many TCM prescriptions to lower its toxicity. Our previous work confirmed
the toxicity of water extract of EK, and explored the detoxic effect of VEK and a combination
use of EK and ZJ using a NMR-based metabonomic approach. In the present study, to further
clarify the toxic and components in water extract of EK, isolation and structure elucidation
were conducted and 8 compounds were obtained. Their structures were identiÀed as triterpenes
Euphol (1), (3ȕ,11ȕ)-3,11-dihydroxylanosta-8,24-dien-7-one (8) and ingenol-typed diterpene esters
3-O-benzoyl-20-deoxyingenol (2), 5-O-benzoyl-20-deoxyingenol (3), 3-O-(2́-E,4́-Z-decadienoyl)20-O-acetylingenol (4), 3-O-(2́-E,4́-E-decadienoyl)-20-O- acetylingenol (5), 20-O-(2́-E,4́-Zdecadienoyl)ingenol (6), 20-O-(2́-E,4́-E- decadienoyl)ingenol (7) by physiochemical means.
Keywords: Euphorbia kansui, traditional Chinese medicine, diterpenes, isolation, metabonomics
1. Introduction
The roots of Euphorbia kansui T. N. Liou ex T. P. Wang (Euphorbiae kansui radix,
Euphorbiaceae), Chinese name Kansui , is one of the very important herbal drugs listed in
Chinese Pharmacopoeia [1a]. As a drastic hydragogue, Euphorbia kansui (EK) possesses medical
functions of removing water retention, subduing swelling and dissolving lumps and so on. It
is reported that the ether, ethyl acetate and ethanol extracts of EK possessed toxicity, and themain toxic components of these extracts are diterpenes [2]. According to the traditional Chinese
medicine (TCM) theory, to reduce the toxicity of EK, vinegar processed EK (VEK) has beencommonly used and the vinegar processing did reduce the toxicity of EK [3].
In the TCM theory, a combination of herbs and constituents usually attribute to synergism− 67 −

therapeutic and pharmacological effects, reduction side effects or toxicities, which termed TCM
compatibility . The compatibility is being received increasing attention at the present time.
The fruits of Zizyphus jujuba (ZJ, Ziziphi fructus), also listed in Chinese Pharmacopoeia [1b]is often prescribed together with EK as a drug pair to lower the toxicity of EK in a number ofprescriptions. It is reported that the triterpenoids of ZJ reduced the toxicity of ethanol extract of
EK [4]. The important thing is, the water extracts of EK or VEK showed no toxicity [3], however,water decoctions of EK or VEK are commonly used in TCM clinic and considered to have toxicity
in TCM. Our previous study shown that when rats were co-administered with water extract of
EK and water extract of ZJ exhibited a quite unique de-toxic effect, toxic symptom of EK was
lower and prior to that administered with water extract of EK vinegar-treated only (Fig. 1).

Fig. 1

In order to understand the Traditional Chinese Medicine Formulations scientifically andclarify how the detoxicant of ZJ interacts with toxicant of EK to reduce side effects, especially theinteractions of components contained in EK and ZJ, we Àrst to isolate the toxicants in the water
extract of kansui.
2. Results and discussion
The water extract of roots of EK was subjected to a series of chromatographic separations
and resulted in the isolation of eight reported compounds: triterpene-type Euphol (1) [5] andingenol-typed diterpene esters 3-O-benzoyl-20-deoxyingenol (2) [6], 5-O-benzoyl-20-deoxyingenol(3) [6], 3-O-(2́-E,4́-Z-decadienoyl)-20-O-acetylingenol (4) [7], 3-O-(2́-E,4́-E-decadienoyl)-20-Oacetylingenol (5) [8], 20-O-(2́-E,4́-Z-decadienoyl)ingenol (6) [8], 20-O-(2́-E,4́-E-decadienoyl)
ingenol (7) [8], (3ȕ,11ȕ)-3,11-dihydroxylanosta-8,24-dien-7-one (8) [9]. The structures of theseknown compounds were determined by analyzing their spectroscopic data and comparison with
reported values in the literature.

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2

HO

2

1
3

O
R1 HO
O
R2

4

1 Euphol

12
1
4

10

11 O
14
8
7

H
15

HH

2 R1 = benzoyl, R2 = H
2

O
R1 HO
O
R2
O
R3
4 R1 =

4'

2'

O

12
1
4

10

11 O

H

14
8
7

HH

5'

3 R1 = H, R2 = benzoyl

15

R2 = H, R3 = Ac

3'HO
R2 = H, R3 = Ac

5 R1 =

1

2O
HO

6 R1 = H, R2 = Ac, R3 = O

3

4

O
8

7 R1 = H, R2 = Ac, R3 =O

Here, take compounds 6 and 7 as examples for the discussion of the structure identiÀcation.
Ingenol-type diterpenes are very difficult to isolate due to their structural similarity especial
the long carbon-chain substitutes which just shown E and Z conÀgurational difference. We got
compounds 6 and 7 from subfraction Fr 10-4 by semipreparitive HPLC with a reverse phasecolumn and normal phase preparative TLC (HPLC spectrum shown as Fig. 2).

Fig. 2
13

C-NMR spectrum of 6 showed 30 carbon atoms, and chemical shifts suggested the presenceof a carbonyl, an ester carbonyl, and four oxygen-bearing carbon atoms that corresponding toIngenol-type diterpenes esters. Meanwhile, most of the diterpenes that have a 2,4-decadienoyloxy− 69 −

group isolated from Euphorbia species possess an E,Z- or Z,E-conÀguration in the double bondsof the ester residue, In compounds with the E,Z-conÀguration [7,10], the coupling constants arenormally J2́,3́ = 15 Hz, J4́,5́ = 11 Hz, and H-3́ appears at about į = 7.68 as a double doublet (J2́,3́= 15 Hz, J3́,4́ = 11 Hz). In compounds with the Z,E-configuration [11], the coupling constants
are normally J2́,3́ = 11 Hz, J4́,5́ = 15 Hz, and the H-3 signal resonated at about į = 7.40, moving
upfield by about 0.3 ppm compared with those with the E,Z-configuration, and appearing as
a double doublet (J 2́,3́ = 15 Hz, J3́,4́ = 11 Hz). In the 1H-NMR spectrum of 6, the H-2́ signal
appeared at į 5.85 as a doublet (J2́,3́ = 15.2 Hz), and the H-3́ signal was found at į 7.60 as a
double doublet (J2́,3́ = 15.2 Hz, J3́,4́ = 11.0 Hz) corresponding to a 2́,3́-trans double bond. Thus,
compound 6 was concluded to be 20-O-(2́-E,4́-Z-decadienoyl)ingenol.

6Fig. 3 1H-NMR(CDCl3) of compound 6

HO

3'

5'
4'

2
1

HO
O HO

4 10
8

O

7

2'

11

12

H

O

14

HH

15

7


1

Fig. 4 H-NMR(CDCl3) of compound 7− 70 −

13

C-NMR and 1H-NMR of compound 7 shown a different pattern from that of an E,Z- or Z,E-

conÀguration in the double bonds of the ester residue. In the case of 7, H-2́ appeared at į 5.78 asa doublet ( J 2́,3́ = 15.2 Hz), corresponding to a 2́,3́-trans double bond. The overlapping signals
at į 6.14, attributed to the H-4́, H-5́ signals, did not offer much information as to the nature of
the double bond. But all the signals marched well with that of reported datas which conÀrmed to
be a E,E-conÀguration in the double bonds of the ester residue. Thus far the structure of 7 wasestablished as 20-O-(2́E,4́E-decadienoyl)ingenol.
It is reported that Euphol (1) together with some other this type of triterpenes [5b] showa moderate cytotoxicity on Àbroblasts, ID50 about 100 ȝg/mL. Wang et al [8] reported that theisolation of compounds 5, 6, and 7 by a bioassay guided method and these diterpenes shown
potent anticancer activity against xenopus cell line at concentration of 0.5 ȝg/mL. There is nobioactivity data of compound 8 reported.
With these compounds in hand, our next step is to assay their cytotoxicity in vitro and scale
up those compounds with potent activity for bioassay in vivo. At the same time identiÀcation ofdetoxicants of the water extract of ZJ will be carried.
3. Experimental Section
3.1 General Experimental Procedures. NMR spectra were taken on a JEOL JNM-LA400
spectrometer with tetramethylsilane (TMS) as an internal standard, and chemical shifts are
expressed in į values. FABMS and HRFABMS measurements were carried out on a JEOL JMS700T spectrometer, and glycerol was used as a matrix. Column chromatography was performed
with normal-phase silica gel (silica gel 60N, spherical, neutral, 40-50 ȝm, Kanto Chemical Co.,
Inc.) and reversed-phase silica gel (Cosmosil 75C18-OPN, Nacalai Tesque Inc.). Medium-pressureliquid chromatography (MPLC) was performed with a Buchi pump module C-650 system.
Preparative TLC was carried out on precoated silica gel 60F254 and RP-18F254 plates (Merck,
0.25 or 0.50 mm thickness).
3.2 Plant Material. The dried roots of Euphorbia kansui L. were collected in Xianyang, Sannxi
Province, People’s Republic of China, in October 1999. A voucher specimen was deposited at theMuseum of Materia Medica, Research Center for Ethnomedicines, Institute of Natural Medicine,
University of Toyama, Japan.
3.3 Extraction and Isolation. The dried roots of E. kansui (1 kg) were extracted twice withwater under reflux. Evaporation of the solvent under reduced pressure from the combined
aqueous solution gave the aqueous extract (200 mL). The extract was extraced with ethyl acetate(3 X 200 mL). The amount extracted was 10 g, and the residual aqueous extract weighed 23 g.
The ethyl acetate fraction was subjected to silica gel column chromatography with MPLC
using an EtOAc-hexane gradient system. The column chromatographic fractions (100 mL each)were combined according to TLC monitoring into 10 subfractions (Fig. 5).

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Water extract 10.0 g
MPLC, eluate: hexane/ethyl acetate,
EA 0% to 100%




Fr 1 

Fr 2

Fr 3

Fr 4

440 mg

140 mg80 mg

1400 mg 500 mg

Fr 5

Fr 6

Fr 7

Fr 8

Fr 9

Fr 10

850 mg

200 mg

570 mg

230 mg1830 mg

Fig. 5

Fr 1 and 2 (580 mg): Combined Fr 1 and 2 was rechromatographed on silica gel with MPLC
using a chloroform-hexane gradient system to afford 32 subfractions. NMR ( 1H, 13C, CDCl3)
indicated that the main component was fatty acid esters and wax.
Fr 4 (1400 mg): Fr 4 was rechromatographed on silica gel with MPLC using a EtOAc-hexane
gradient system to afford a white crystal (1, 952 mg) as the main component.
Fr 5 and 6 (1350 mg): Combined Fr 5 and 6 was rechromatographed on silica gel with MPLC
using a chloroform-hexane gradient system to afford 25 subfractions. NMR ( 1H, 13C, CDCl3)
indicated that the main component was fatty acids.
Fr 7 (200 mg): Fr 7 was subjected to normal-phase preparative TLC (NPTLC) with Hexane/
CHCl3/EtOAc (5:12:1) afford 7 subfractions (Fig. 6). Further separation of Fr 7-4 (30 mg) bysemipreparitve HPLC (Waters Discovery C18 column, 10 X 250 mm, 5 ȝM, MeOH-H2O, 96:4, Áowrate 2 mL/min; UV detector set at 211 or 254 nm) based on TLC, afford 7-4-1 (1.4 mg, Rt 10.9min) and 7-4-2 (2.4 mg, Rt 12.3 min), NMR (1H, 13C, CDCl3) conÀrmed that 7-4-2 was a mixtureof two ingenol-typed diterpene esters.

Fr 7, 200 mg


NPTLC, eluate:
Hexane/Chloroform/Ethylacetate,5:12:1







Fr 7-1

Fr 7-2

Fr 7-3

Fr 7-4

Fr 7-5

Fr 7-6

Fr 7-7

5.7 mg

8.0 mg17.0mg30.0 mg40.0 mg

13.0 mg

9.0 mgFig. 6

Fr 8 (570 mg): Fr 8 was subjected to normal-phase preparative TLC with MeOH/CHCl3/ (1:9)
afford 8 subfractions (Fig. 7). Further separation of Fr 8-0 (110 mg) by semipreparitve HPLC
(Waters Discovery C18 column, 10 X 250 mm, 5 ȝM, MeOH-H2O, 88:12, Áow rate 2 mL/min; UV− 72 −

detector set at 211 or 254 nm) based on TLC, afford Fr 8-0-1 (5.4 mg, Rt 9.5 min) and 8-0-2 (5.4mg, Rt 11.2 min), but NMR (1H, CDCl3) conÀrmed that each of them is not pure compound, there
contain two isomers. Combined Fr 8-0-1 and 8-0-2 further puriÀed by NPTLC (hexane/EA, 4:1),
furnished compounds 8-0-2-1 (2, 3.3 mg) and 8-0-2-2 (3, 3.3 mg), which were characterized by
NMR (1H, 13C, and 1H-COSY, CDCl3).

Fr 8, 570 mg


NPTLC, eluate:
Methanol/Chloroform, 1:9Fr 8-0

Fr 8-1

Fr 8-2

Fr 8-3

Fr 8-4

Fr 8-5

Fr 8-6

Fr 8-7110.0 mg

13.0 mg24.0 mg41.0 mg

13.0 mg

9.0 mg

4.0 mg

4.0 mgFig. 7

Fr 9 (230 mg): Separation of Fr 9 by semipreparitve HPLC (Waters Discovery C18 column,
10 X 250 mm, 5 μ M, MeOH-H2O, 92:8, Áow rate 2 mL/min; UV detector set at 211 or 254 nm)
afford 9-1 (< 0.2 mg, Rt 3.2 min), 9-2 (1 mg, Rt 7.9 min), 9-3 (5.5 mg, Rt 10.0 min), 9-4 (4, 8.7 mg,
Rt 12.9 min), and 9-5 (5, 4.0 mg, Rt 13.8 min), which were characterized by NMR (1H, 13C, CDCl3)
and compared with Ref.
Fr 10 (900 mg): Fr 10 was rechromatographed on reverse-phase silica gel with MPLC using aacetone-water gradient system to afford 8 subfractions (Fig. 8) based on TLC. Further separationof Fr 10-4 (120 mg) by semipreparitve HPLC (Waters Discovery C18 column, 10 X 250 mm, 5 ȝM,
MeOH-H2O, 85:15, Áow rate 2 mL/min; UV detector set at 211 or 254 nm) based on TLC, afford
10-4-1 (4.1 mg, Rt 13.5 min), 10-4-2 (6, 10 mg, Rt 17.5 min), 10-4-3 (9.0 mg, Rt 19.0 min), 10-4-4(11.7 mg, Rt 22.5 min), 10-4-5 (2.7 mg, Rt 25.5 min). NMR (1H, 13C, CDCl3) conÀrmed that except
10-4-2 was pure other four fractions were mixtures of ingenol-typed diterpene. Further puriÀed
10-4-3 by NPTLC (CHCl3/EA, 5:1) furnished 10-4-3-1 (7, 3.5 mg). Further purified 10-4-4 by
NPTLC (CHCl3/EA, 5:1) furnished 10-4-4-1 (1.4 mg), 10-4-4-2 (8, 1.5 mg), 10-4-4-3 (1.4 mg).
Fr 10, 900 mg


RP-MPLC, eluate:acetone/water, 20 - 100%







Fr 10-1

Fr 10-2

Fr 10-3

Fr 10-4

Fr 10-5

Fr 10-6

Fr 10-7

Fr 10-8

60 mg

83 mg

90 mg

120 mg

150 mg

123 mg

150 mg

100 mgFig. 8− 73 −

Acknowledgements
This work was supported in part by Grant-in-aid for 2011 general research II from Instituteof Natural Medicine, University of Toyama, Japan. Author is very grateful to professor Shigetoshi
Kadota and associate professor Yasuhiro Tezuka (Institute of Natural Medicine, University of
Toyama, Japan ) for offer good platform for this research. And I thank Dr. Feng Li (Institute of
Natural Medicine, University of Toyama, Japan ) for his technical assistance.
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