Bulletin of the Transilvania University of Bra ov
Series VI: Medical Sciences • Vol. 4 (53) No. 2 - 2011
CONTRIBUTIONS TO THE
PHYTOCHEMICAL STUDY OF SOME
SAMPLES OF AJUGA REPTANS L. AND
AJUGA GENEVENSIS L.
G. GHITA1,2,3 O. CIOANCA3 E. GILLE2
R. NECULA2 M. M. ZAMFIRACHE1 U. STANESCU 3
Abstract: Continuing a series of investigations regarding the chemical
variability of some vegetal species, we initiated a comparative chemical
study of the Ajuga reptans L. and Ajuga genevensis L. species used in
Romanian folk medicin. We investigated the iridoidic, flavonoidic fractions
as well as those of the polyphenolcarboxylic acids with the help of such
investigation techniques as: thin layer chromatography, spectrophotometry
and high performance liquid chromatography. The analysed vegetal material
was made up of the aerial parts of the two Lamiaceae species, prelevated
from individuals belonging to some natural populations from the North of
Moldavia. We could notice that Ajuga reptans is richer in antimflamatory,
immunomodulating and hepatoprotecting iridoids than Ajuga genevensis,
while the second has a slightly higher content of antioxidant and diuretic
polyphenols. In the same time, we could notice the existence of an
interspecific variability due to some genetic causes, but also intraspecific, of
a pedo-climatic nature. By means of HPLC we proved the presence in the
studied vegetal material of chlorogenic and caffeic acid, of apigenol and
luteolin-7-O-glucosyde.
Key words: Ajuga reptans L., Ajuga genevensis L., chemical variability,
iridoids, polyphenols.
Introduction
Ajuga reptans L. is aplant used in the
traditional medicine of many countries
from the centre and, especially, the eastern
part of Europe. The extracts obtained from
bugle (Ajuga reptans L.) are used due to
the content of polyphenols of the
flavonoidic and polyphenolcarboxylic
1
acids type (due to its antioxidant, vascular
and antimicrobial qualities), as well as of
iridoids (antiinflamatory and wound
healing) as antidiarrhoeaic, antileucoreic,
hepatoprotecting and vulnerar. Unlike
bugle, blue bugleweed, Ajuga genevensis
L., is used only in our country as a
substitute of the medicinal species, as, due
to the fact that it is not very demanding as
Faculty of Biology, “Al. I. Cuza” University, Carol I 22, 700505, Iasi.
NIRDBS /„Stejarul” Biological Research Centre, Alexandru cel Bun 6, 610004, Piatra Neamt.
3
Faculty of Pharmacy, "Gr. T. Popa" University of Medicine and Pharmacy, Universitatii 16, 700115, Iasi.
2
8
Bulletin of the Transilvania University of Bra ov. Series VI • Vol. 4 (53) No. 2 - 2011
to the pedoclimatic conditions, it is largely
outspread in the wild flora.
In our researches, we have comparatively
analysed the chemical composition of four
samples Ajuga reptans L. And five of
Ajuga genevensis L. harvested at the
beginning of May 2010 in the north eastern
part of Moldavia.
The objective of the investigation was
to establish the chemical similarities and
differences between the two species
especially regarding the composition of the
irridoidic, flavonoidic and polyphenolic
acid fractions.
The aims of the phytochemical study
were to determine the biosynthetic level of
the
flavonoidic,
iridoidic
or
caffeic/chlorogenic acid type derivates, as
well as the influence of some genetic
factors (the belonging of the species to the
same genus) or environmental (soil and
climate), particular and different, also
depending on the location of the analysed
natural population. Our studies were
performed on the vegetal material
harvested in 2010.
2. Material and Method
The vegetal material of Ajuga reptans L.
and Ajuga genevensis L. samples harvested
at the beginning of May 2010 from natural
populations identified in the north of
Moldavia was dried at room temperature
and extracted with absolute methanol
(DER=0.5:100g/mL),
at
warm
temperature, till exhaustion. These extracts
served to achieve the spectrophotometric
determinations of polyphenols and
iridoids.
The TLC and HPLC analyses were
performed on extracts for which the ratio
drug/extract (DER) was of 3:100 g/mL.
The analysed samples were prelevated due
to the data in table 1.
Table 1
The origin of the vegetal material (2010)
Sample
code
AR1
AR2
AR3
AR4
AG1
AG2
AG3
AG4
AG5
Location
Altitude Collection
(m)
date
Ajuga reptans L 2010
Guranda
171
04.05
Baisa
282
04.05
Bicaz-Baraj
560
06.05
Potoci
648
06.05
Ajuga genevensis L 2010
Albesti
70
04.05
Draslea
100
04.05
Stanceni
176
04.05
Baisa
282
04.05
Grozavesti
530
04.05
To get an idea of the iridoidic and
triterpenic spectrum existing in the
prelevated plants, we initially achieved a
TLC study using the exhaustings with
DER 3:100 g/mL [2].
The spectrophotometric determinations aimed the biosynthetic level of the
above mentioned secondary metabolites, the
analysed methanolic extracts having the
DER 0.5:100 g/mL. In these extracts we
determined the flavonoids by a treatment
with aluminium chloride when the bioactive
components form internal complexes with
Al3+, intensly yellow coloured, for which the
extinction was read on the spectrophotometer
at λ=413nm, using luteolin as standard [3].
The polyphenolic acid dosing was performed
by
treating
the
extracts
with
phosphowolframic acid in an alkaline
medium, when we obtained blue colourings,
colourimetered at λ=660nm, compared to the
chlorogenic standard [3]. The iridoids
extracted from the vegetal product and
purified by passing the extract over a
silicagel column, were treated with PABA in
acid medium, when, at warmth, there forms a
blue coloured compound to be colourimetered at λ=590 nm, the standard curve
being traced with aucuboside [4, 5, 6, 7]. The
methanolic exhaustions for which DER was
equal to 3:100g/mL were also qualitatively
G. GHITA et al.: Contributions to the Phytochemical Study of some Samples …
and half-quantitatively analysed by HPLC
for polyphenols.
Chromatography conditions: an HPLC
Agilent 1100 system with registration and
integration of the chromatograms by
means of the Borwin computerized system
and multidiode detector.
The stationary phase: eclipse XDB-C18
(150mm x 4,6mm; 5 m); The mobile
phase: component A=sodium acetate 2mM
brought at the pH=2.5 with acetic acid;
component B=acetonitryl; gradient: 98%
A;2% B (0-20 min.); 86% A:14%B (20-40
min.); 80%A:20% B (40-50 min);
70%A:30B (50-60 min.); 98%A:2%B
(after 65 min.); flow: 1mL/min.;
temperature in the column compartment
250 C; detection: UV 320nm for samples
and standards; in addition for each
standard we registered the absorbtion
9
spectrum in the interval of 200-400nm;
applied volume: 100 L fir the solutions to
be analysed and the standard.
Results and Discussions
The important part of our study was the
comparative phytochemical analysis of the
two Ajuga species to draw conclusions
regarding the inter and intraspecific
chemical variability. To monitorize the
similarities and differences of chemism in
the triterpenic and iridoidic fractions, we
analysed the two methanolic Ajuga
exhausts by the TLC method. In fig.1 and
2 are the thin layer chromatograms for the
terpenes and iridoids of the Ajuga reptans
and Ajuga genevensis samples.
Legend: samples- v. tab.1;
standards: β-sitosterol (B), stigmasterol (S), oleanolic acid (O), ursolic acid (U)
Fig. 1. TLC for terpenes of „Ajuga reptans” and „Ajuga genevensis” samples
Legend: samples-v. tab.1;
standards: aucuboside (Au), harpagide (Hp), 8-O-acetyl harpagide (8-Hg)
Fig. 2. TLC for iridoids of „Ajuga reptans” and „Ajuga genevensis” samples
Bulletin of the Transilvania University of Bra ov. Series VI • Vol. 4 (53) No. 2 - 2011
10
Regarding the iridoids identified in the
Ajuga extracts, they seem to be represented
by two components in both species, out od
which the inferior one (Rf=0.53) seems to
be harpagide, and the second remaining
unknown due to the lack of the
corresponding stamdard. A third spot,
superior but slight, appears in the case vof
two Ajuga reptans samples (AR3 and
AR4) and one Ajuga genevensis (AG5) and
seems to belong to the 8-O-acetyl
harpagide.
If from TLC study produced the main
conclusion was the confirmation of the
similar chemism in the same species, the
highlighting of the differences between the
two
species
respectively,
the
spectrophotometric dosings
of the
flavonoids, polyphenolic acids and iridoids
produced
differenciated
quantitative
assertions. In tablel 2 we present the results
obtained
by
the
quantitative
determinations.
Table 2
The content in iridoids and polyphenols dosed in vegetal material (Ajuga reptans
L./Ajuga genevensis L.) prelevated from different natural populations in 2010
Sample Code
Origin
Determination in 100g herba
Iridoids
Flavonoids
Polyphenolic acids
g aucubozid %
g luteolin % g chlorogenic acid %
Ajuga reptans L.
AR1
AR2
AR3
AR4
Guranda
Baisa
Bicaz – Baraj
Potoci
1.329 ± 0.0097
1.078 ± 0.0032
1.983 ± 0.0010
1.673 ± 0.0085
0.501 ± 0.0034
0.471 ± 0.0047
0.455 ± 0.0066
0.563 ± 0. 0019
1.581 ± 0.0022
1.690 ± 0.0016
1.870 ± 0.0013
1.744 ± 0.0031
Ajuga genevensis L.
AG1
AG2
Albesti
Draslea
0.988 ± 0.0059
0.8763 ± 0.0036
AG3
AG4
AG5
Stauceni
Baisa
Grozavesti
0.641 ± 0.0071 0.561 ± 0.0093 1.825 ± 0.0002
1.033 ± 0.0081 0.417 ± 0.0055 1.593 ± 0.0035
0.9512 ± 0.0099 0.545 ± 0.0043 1.733 ± 0.0006
As may be noticed, the Ajuga reptans
samples are richer in immunomodulating
and intiimflamatory iridoids, while in case
of Ajuga genevensis the antioxidant
polyphenol content is higher than in the
case of bugle.
If we graphically represent the values
from the table, we will notice that
regarding the iridoids (fig. 3) the richest
sample proved to be Ajuga reptans
hervested at Bicaz-Baraj, the other bugle
samples having a lower content.
Compared to this
intraspecific
variability determined for the iridoidic
fraction of bugle, Ajuga genevensis
0.698 ± 0.0012
0.839 ± 0.0011
2.020 ± 0.0074
1.876 ± 0.0061
presents a similar situation, yet this time
the highest non-volatile monoterpen
content being found in the Baisa sample,
and the most reduced in that of Stauceni.
On the other hand, it is obvious that
along with the intraspecific variability,
there also is a genetic, interspecific,
variability, Ajuga reptans being much
richer in iridoids than blue bugleweed.
Regarding the flavonoids, (fig. 4), we
notice that the richest sample in such
compounds is that of Ajuga genevensis
from Draslea, Ajuga reptans containing
smaller flavonoid quantities.
G. GHITA et al.: Contributions to the Phytochemical Study of some Samples …
11
Fig. 3. The graphic expression of the iridoid content determined in the
„Ajuga” exhausts (2010)
2.5
g% luteolin
2
1.5
1
0.5
0
AR1 AR2 AR3 AR4
AG1 AG2 AG3 AG4 AG5
Fig. 4. The graphic expression of the flavonoid content determined in
the ”Ajuga” exhausts (2010)
Comparing the values of the Baisa Ajuga
reptans with those of Ajuga genevensis of
the same location, we notice that the
quantitative differences in case of the three
fractions of active principles are not major
(fig. 5), for the same location, the climatic
and soil conditions beinf identical for the
two species.
Bulletin of the Transilvania University of Bra ov. Series VI • Vol. 4 (53) No. 2 - 2011
12
Fig. 5. The graphic expression of the iridoid and polyphenol content
determined in methanolic extracts (DER 0,5:100) of bugle and blue bugleweed
harvested in the same location-Baisa-in 2010
To better appreciate the similaritiesa and
differences of the polyphenolic spectrum
of the nine Ajuga samples, we resorted to
the HPLC analysis. In table 3 we present
the components as well as the
corresponding quantities identified the
polyphenols
in
exhausts
(DER 3:100 g/mL).
Table 3
Polyphenols identified in Ajuga reptans and Ajuga genevensis methanolic exytracts
(2010)
Sample
code
Chlorogenic
Caffeic
Luteolin 7-OApigenol
acid
acid
glucoside
ug/mL mg/100g ug/mL mg/100g ug/mL mg/100g ug/mL mg/100g
Ajuga reptans L
AR1
AR2
AR3
AR4
3.62
1.52
2.97
2.03
12.08
5.07
9.90
6.77
0.97
0.97
1.23
1.32
3.25
3.25
4.10
4.39
0.35
0.14
0.09
0.15
1.17
0.48
0.31
0.84
1.43
0.63
3.07
1.30
4.76
2.11
10.25
4.33
1.72
3.15
3.32
5.16
5.10
1.49
3.90
2.83
3.15
3.94
4.98
12.99
9.43
10.50
13.15
Ajuga genevensis L
AG1
AG2
AG3
AG4
AG5
4.21
9.73
6.01
44.68
7.08
14.05
32.44
20.05
148.93
23.61
1.06
1.60
1.73
0.83
2.07
3.55
5.34
5.77
2.77
6.90
0.51
0.94
1.00
1.55
1.53
G. GHITA et al.: Contributions to the Phytochemical Study of some Samples …
Calculating the concentrations of the
chlorogenic acids, caffeic acids, luteolin-7O-glucoside and apigenol existing in the
100 g of vegetal product, we obtained the
13
values indicated by the same table 3.
Expressing these concentrations graphically,
we have the situation from fig. 6.
Fig. 6. The variation of the content in chlorogenic acid, caffeic acid, luteolin-7-Oglucoside and apigenol (g/100g herba) determined by HPLC in the samples of „Ajuga
reptans” and „Ajuga genevensis” (2010)
One can notice that of the two
poliphenolic acids, the chlorogenic one is
quantitatively better represented, there
appearing, as already seen, great
intraspecific variations, yet espacially
interspecific. The second poliphenolic
acid, the caffeic acid has a smaller
concentration, still the Ajuga genevensis
are richer in this component. Coming back
to the achieved spectrophotometric
determinations, we will notice that the
majority of the components of the group
were not identified. Thus, we have the
question of how many more, not identified
acids, are there in the vegetal products.
Analogous, we notice that out of the
flavonoidic fraction, we could identify only
two components: apigenol and luteolin-7-Oglucoside. In this case, apigenol dominates,
yet we again notice that the Ajuga genevensis
14
Bulletin of the Transilvania University of Bra ov. Series VI • Vol. 4 (53) No. 2 - 2011
populations seem to be richer in this flavonic
aglicon than those of Ajuga reptans. Again,
there arises the question of what flavonoids
coexist in Ajuga genevensis L. and Ajuga
reptans L.
Conclusions
The comparative phytochemical study
on the two Ajuga species, Ajuga reptans
and Ajuga genevensis aimed to highlight
the qualitative and quantitative variations
of the iridoidic, flavonoidic and
polyphnolic acids fractions in dry vegetal
material. We noticed that, in both species,
the individuals originated from populations
developed in different locations, present
variations of larger or more restricted
limits especially quantitative and less
qualitative for the same group of secondary
metabolites.
Ajuga reptans proved to be richer in
iridoidic components of the harpagide type
(antiinflamatory, hepatoprotecting and
immunomodulating)
while
Ajuga
genevensis is slightly richer in polyphenols
(antioxidant and diuretic). Out of the
polyphenolic components, by HPLC, we
identified and quantified chlorogenic acid,
caffeic acid, apigenol and luteolin-7-Oglucoside.
Aknowledgement
The paper was presented at the: al IV lea
Simpozion National de Etnofarmacologie
cu participare internationala cu tema
"Etnofarmacologia la interfata bioalimentfitomedicament" - iunie 2011 Brasov –
Sirnea.
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