STUDIA UNIVERSITATIS BABEŞ-BOLYAI, PHYSICA, SPECIAL ISSUE, 2001
THE DETERMINATION OF THE THERAPEUTIC COMPOUNDS
FROM Aristolochia Clematitis BY GC/MS
R. PODEA, M. CULEA, L. FROMONDI
S.C. Natex s.r.l. Sos. Cluj-Oradea km8 , P.O. Box 374 3400 Cluj-Napoca
SUMMARY. Some extracts of the medicinal plant Aristolochia Clematitis from Romania
have been investigated by GC-FID and GC/MS to identify compounds responsible for its
therapeutical properties. Three kinds of extracts were investigated.
The tincture was prepared by mixing all parts of the plant (roots, steams, leaves) with an
50% alcoholic solution for 30 days. The infusion was also prepared by mixing all parts of
the plant with hot water for 20 min. These two extraction methods were compared for
extraction of the active therapeutic compounds of Aristolochia Clematitis.
In parallel, another study was performed to identify the distribution and the
concentration of the active compounds in the roots, steams and leaves of this plant. For this
purpose we have prepared three alcoholic (96 % alcoholic solution) extracts from each part
of the plant and we have studied them separately.
Introduction
Aristolochia Clematitis, a very toxic herb, member of the Aristolochiaceae
family, popularly named Marul Lupului, is a rare medicinal plant which has a very long
history of medicinal use, though it is little used by present-day herbalists. It is an
aromatic tonic herb that stimulates the uterus, reduces inflammation, controls bacterial
infections and promotes healing. The flowering herb, with or without the root, is
abortifacient, antiinflammatory, antispasmodic, diaphoretic, emmenagogue, febrifuge,
oxytonic and stimulant. The plant contains a complex of acids so called “aristolochic
acids” which stimulate white blood cell activity and speeds the healing of wounds if it
is used in correct concentration. Aristolochic acid is active in a concentration of
1:100000. Externally it is used in the treatment of slow-healing cuts, eczema, infected
toe and finger nails etc., but internal consumption can cause damage to the kidneys and
uterine bleeding.
The objective of this work is to identify and characterise the compounds of
therapeutic value extracted from Aristolochia Clematitis. The analytical methods
chosen are gas-chromatography (GC-FID) and gas-chromatography/mass spectrometry
(GC/MS). The methods were applied to characterise the infusion and tincture prepared
from this plant and to make a comparison between the alcoholic extracts of the roots,
stems and leaves.
Experimental
Aristolochia Clematitis plants were harvested in July 2001.
Extraction procedure
For the preparation of tincture and infusion we have used air dried, crushed
and mixed plants, and for the alcoholic extract (96% alcoholic solution) fresh roots,
THE DETERMINATION OF THE THERAPEUTIC COMPOUNDS FROM Aristolochia Clematitis BY GC/MS
steams and leaves. The tincture was prepared by mixing all parts of the plant (roots,
steams, leaves) with an 50% alcoholic solution for 30 days. The infusion was also
prepared by mixing all parts of the plant with hot water for 20 min, and the alcoholic
extracts by mixing the fresh parts of the plant with a 96% alcoholic solution for 12
days.
Sample preparation for GC-FID and GC/MS
3 ml of tincture were mixed for 5 min with 3 ml distilled water, pH 3, 0.5g
NaCl and 1 ml solvent A (ethyl acetate:hexane:methylen chloride 5/1/1, v/v/v). Then
the organic phase was collected and 4 µl were injected in GC/MS.
6 ml of infusion, pH 3 and 0.5g NaCl were mixed for 5 min with 1 ml solvent
A and 4 µl of the organic phase were injected in GC/MS.
3 ml of 96 % alcoholic extracts were mixed for 5 min with 3 ml distilled water,
pH 3, 0.5g NaCl and 1 ml solvent A. Then the organic phase was collected and 4 µl
were injected in GC-FID.
GC-FID Analysis
GC-FID analysis was performed with a Hewlett Packard GC system 6890 with
FID. Compounds were separated on a 30m x 0.25mm HP-5 fused-silica capillary
column coated with 0.25µm film.
The column was maintained at 50C for 2 min, then it was programmed at
8C/min to 250C, then 30C/min to 310C which was maintained for 10 min. Helium
was used as carrier gas with a flow rate of 1ml/min and split ratios 1:20. The injector
and detector temperatures were 250C.
GC-MS Analysis
GC-MS analysis was performed with a Hewlett Packard GC 5890 coupled with a
MS engine 5989B in the EI mode. Compounds were separated on a 30m x 0.25mm HP5MS fused-silica capillary column coated with 0.25µm film. The column was maintained at
50C for 2 min after injection, then programmed at 8C/min to 250C, then 30C/min to
310C which was maintained for 10 min. Split ratio was 1:5 and helium flow rate 1ml/min.
Injector, interface, ion source and quadrupol analyser temperatures were 250C, 280C,
200C, 100C, respectively. The mass spectrometer was operated in electron-impact (EI)
mode; electron energy was 70 eV and electron emission 300 A. Compounds were
identified using the Wiley, NBS and NIST library spectra.
Results and discussion
Use of GC/MS (Fig.1), enabled identification of the most components in both
samples tincture and infusion. The compounds identified are listed in Table 1. Some
contaminants in as dibutylphtalate, tR 28.3min were neglected.
The predominant components of the tincture are terpenic compounds, fatty
acids esters and aristolochic acid derivatives. We have studied the mass spectra of
379
R. PODEA, M. CULEA, L. FROMONDI
compounds no. 33 (M=294), 34 (M=293) and 35 (M=355) mass spectra and we have
concluded that these are some aristolochic acid derivatives (Fig.2,a,b,c). The
derivatives of the aristolochic acid seem to be present in root and stems of this plant.
The derivatives have the same fragment ions as aristolochic acid ( Fig. 2,d) as: M-15,
M-31, M-43 in the case of the first two, or M-46 in the case of the last derivative. m/z
294 seems to be the molecular ion of a derivative of aristolochic acid by loosing 0=N-OH
radical. The second derivative has m/z 293, as molecular ion containing odd number of
nitrogen in the molecule. The third derivative has m/z 355 as molecular ion which
represent m/z 341 (molecular ion of aristolochic acid ) + 14.
a)
T
A
b
u
n
d
a
n
c
e
1
0
0
0
0
0
9
5
0
0
0
9
0
0
0
0
8
5
0
0
0
8
0
0
0
0
7
5
0
0
0
7
0
0
0
0
6
5
0
0
0
6
0
0
0
0
5
5
0
0
0
5
0
0
0
0
4
5
0
0
0
4
0
0
0
0
3
5
0
0
0
3
0
0
0
0
2
5
0
0
0
2
0
0
0
0
1
5
0
0
0
1
0
0
0
5
0
0
T
i
m
e
-
-
I
C
:
M
L
2
.
D
0
0
0
>
5
.
0
10
0
.
0
1
0
5
.
0
2
0
0
.
0
2
0
5
.
0
3
0
0
.
0
3
0
5
.
0
0
b)
mmmmmmm
Abundance
TIC: ML1.D
110000
100000
90000
80000
70000
60000
50000
40000
30000
20000
10000
Time--> 5.00
10.00
15.00
20.00
25.00
30.00
Fig. 1. The ion chromatograms obtained by GC/MS analysis for tincture(a) and
infusion (b)
380
THE DETERMINATION OF THE THERAPEUTIC COMPOUNDS FROM Aristolochia Clematitis BY GC/MS
Table 1
Composition of the tincture and infusion
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Compounds
alpha-pinene
camphene
beta-myrcene
limonen
eucalyptol
camphor
terpinen-4-ol
4-vinyl phenol
bornyl acetate
4-vinyl-2-metoxyphenol
dihydroactinidiolide
caryophyllene oxide
loliolide
benzyl salicylate
1-octadecanol
ethyl hexadecanoate
phytol
ethyl linoleate
octadecatrienoic acid, ethyl ester
M=297
M=294
M=293
M=355
tR
6.1
6.4
6.96
8
8.04
10.3
10.9
11.7
12.8
13.3
16.9
17.6
20.3
21.5
22.8
22.8
24.4
24.8
24.9
29.3
30.9
32.7
33.3
Tincture
%MS
Infusion
%MS
0.89
1.60
0.44
1.13
1.44
3.43
0.00
0.00
2.04
0.00
0.00
2.11
0.00
0.00
2.11
24.40
7.84
8.48
31.87
3.18
13.97
8.16
0.00
0.00
0.00
0.00
3.03
0.00
0.00
7.62
97.58
0.00
13.02
2.22
0.00
16.85
4.84
0.00
0.00
0.00
0.00
0.00
26.14
57.77
12.97
3.13
tR=the retention time
a)
A
S c a n
b u n d a n c e
1 2 0 8
(3 0 . 8 8 9
m in ) :
M
L 1 . D
(-)
2 9 4
2 7 9
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
1 3 7
1 4 7
2 0 0 0
1 0 0 0
m /
0
z -->
3 9
3 0
1 2 5
6 8
6 3 8 7
1 1 1
2 5 1
1 6 5
1 9 5
2 2 3
1 9 3
2 3 5
2 6 5
4 06 08 0
1 01
021
041
061
082
002
022
042
062
083
00 0
381
R. PODEA, M. CULEA, L. FROMONDI
b)
b Abundance
Scan 1284 (32.643 min): ML1.D (-)
1600
1500
1400
1300
1200
1100
1000
900
800
700
600
500
400
300
200
100
m/z-->0
278
293
146
124
51
30
40
75
63
60
80
164
139
87
250
111
193
194
222
100 120 140 160 180 200 220 240 260 280 300
c)
S
b
u
c
n
d
a
a
n
n
1
c
3
e
1
4
(
3
3
.
3
3
5
m
i
n
)
:
M
3
4
0
0
3
8
0
3
6
0
3
4
0
3
2
0
3
0
0
2
8
0
2
6
0
2
4
0
2
2
0
2
0
0
1
8
0
1
6
0
1
4
0
1
2
0
1
0
0
8
0
6
0
4
0
2
2
3
2
2
1
6 5
5
-
382
5
1
1 1
2 1
1
5
5
5
1
1
6
1
5
9
3
1
0
-
0
>
2
4
0
6
0
8
0 1
0 1
0
1
2
0
1
4
0
1
6
0
9
2
8
0
1
2
0
0
2
2
0
2
4
0
2
6
0
3
8
0
.
9
8
1
z
7
1
4
3
8
/
9
L
9
2
5
m
0
3
0
0
3
2
0
4
0
0
D
(
-
)
THE DETERMINATION OF THE THERAPEUTIC COMPOUNDS FROM Aristolochia Clematitis BY GC/MS
d)
Fig. 2
The components of the infusion differ from those found in tincture except
aristolochic acid derivatives. The concentrations (in %MS) of these derivatives in
infusion and tincture are very closed, maybe that is why in traditional medicine are
used both types of extracts with success.
The differences between the compounds that we have found in the roots,
steams and leaves of Aristolochia Clematitis were studied by GC-FID. This study was
performed on the alcoholic extracts of the three parts of the plant. From this study we
have concluded that the compounds found in the root and steam are very similar. The
aristolochic acid derivatives are present in both extracts, but in the leaves these
derivatives are in very low concentration.
Conclusion
The analytical methods used, GC/MS and GC-FID are suitable for medicinal
herb organic compounds determination. The sample preparation method is rapid and
precise.
There is a difference between the compounds extracted from herb by infusion
and tincture but the important thing is that the aristolochic acid derivatives are present
in both of them. On the other side the study shows that their concentration is higher in
the roots and steams. In the leave extracts aristolochic acid derivatives and vitamin F
(polyunsaturated fatty acids) are very low or are absent. In conclusion terpenic
383
R. PODEA, M. CULEA, L. FROMONDI
compounds, fatty acids, coumarins, alkaloids and especially aristolochic acid
derivatives are responsible for the therapeutic activity of this plant.
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