plants
Article
Scopolia carniolica var. hladnikiana: Alkaloidal Analysis and
Potential Taxonomical Implications
Karsten Fatur * , Matjaž Ravnikar
and Samo Kreft
Department of Pharmaceutical Biology, Faculty of Pharmacy, University of Ljubljana, 32 Tržaška cesta,
1000 Ljubljana, Slovenia; Matjaz.ravnikar@ffa.uni-lj.si (M.R.); samo.kreft@ffa.uni-lj.si (S.K.)
* Correspondence: karsten.fatur@gmail.com
Abstract: The present research sought to compare the content of hyoscyamine/atropine and scopolamine in Scopolia carniolica and its contested variety, S. carniolica var. hladnikiana, with the aim of
investigating differences that may be of taxonomical significance. A multi-phase liquid extraction
and high-performance liquid chromatography were used to extract and analyse these alkaloids
in different organs from plants collected over two years at three sites. Our results showed that
hyoscyamine was almost twice as prevalent as scopolamine across our 87 samples. The differences
between organ types were large, but so too were intra-organ differences; differences due to organs
proved to be significant for hyoscyamine, while they were only marginally significant for scopolamine. The collection site also proved to have a significant influence, but only on hyoscyamine
content. The year of collection and the variety proved to not be significant. Our results support the
theory that these two varieties are likely one, a view argued by many others, though more work is
needed to draw concrete taxonomical conclusions.
Citation: Fatur, K.; Ravnikar, M.;
Keywords: Scopolia carniolica; Scopolia carniolica var. hladnikiana; taxonomy; secondary metabolites;
tropane alkaloids; Slovenia; Solanaceae
Kreft, S. Scopolia carniolica var.
hladnikiana: Alkaloidal Analysis and
Potential Taxonomical Implications.
Plants 2021, 10, 1643. https://
doi.org/10.3390/plants10081643
Academic Editor: Cecilia Cagliero
Received: 7 July 2021
Accepted: 3 August 2021
Published: 10 August 2021
Publisher’s Note: MDPI stays neutral
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Licensee MDPI, Basel, Switzerland.
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conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Named after the famed botanist, Franc Hladnik, who first discovered it, Scopolia
carniolica var. hladnikiana, otherwise known as Hladnik’s Scopolia is a plant from the
Solanaceae family that is endemic to a small number of discontinuous sites throughout the
south eastern European country of Slovenia [1,2]. While the common S. carniolica has a
distribution spanning throughout central and southeast Europe, the Hladnik variety (as of
2013) grows in only 10 confirmed sites [3]. Both varieties tend to grow in ravines within
deciduous forests, especially those with high numbers of Fagus spp. [4,5]. The Hladnik
variety is found growing in the presence of the normal variety.
These glabrous plants are small, deciduous perennials, reaching about 20–60 cm in
height and appearing in early spring [4]. Growing up from the rhizomatic rootstock, the simple, ovate leaves (~10–20 × 5–8 cm) are found on short petioles alternately arranged on the
stem and have simple to slightly crenate margins [4]. Campanulate flowers (15–25 mm) with
floral parts of five, bloom in early spring from the leaf axils on comparably long pedicels and
mature into capsules [4].
The primary distinction between S. carniolica and the Hladnik variety is that of the
flower colour; while the standard variety has flowers with a deep violet colour on the
outside of the corolla and a yellow colour on the inside, the Hladnik variety is a yellowgreen throughout [2,4]. That being said, many other differences have been stated at various
points in time, such as the Hladnik variety having shorter leaves, larger flowers, a later
flowering time, and fewer flowers, while the regular variety is said to have purple leaf
veins that are absent in the Hladnik variety [2,4]. However, these differences have largely
proven to be inconsistent, with flower colour being the primary means of differentiating.
Plants 2021, 10, 1643. https://doi.org/10.3390/plants10081643
https://www.mdpi.com/journal/plants
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It is now debated by many whether the Hladnik version is in fact a true variety or if it falls
within the normal variation for S. carniolica.
Where previous investigations into the divide between S. carniolica and the Hladnik
variety were morphologically focused, a comparative analysis of alkaloids has yet to
be performed to see if there are any clear differences in the chemical makeup of these
two plants.
Along with other close relatives in the Solanaceae, S. carniolica contains the tropane
alkaloids scopolamine and hyoscyamine (which racemises to atropine when dried), which
found extensive use throughout the years as a result of their multitude of effects in the
human body [6–8]. Synthesised in the roots and then spread throughout the other organs,
these alkaloids serve to protect the plants from predation as well as from various other
forms of life and pathogens [9,10].
This research sought to examine the levels of the alkaloids hyoscyamine/atropine and
scopolamine in samples of both S. carniolica and S. carnolica var. hladnikiana, comparing
variety, organ, year of collection, and collection site to determine if there is any significant
difference in alkaloid levels between the two plants that would support their division into
separate varieties.
2. Results and Discussion
Through our analysis of standards of atropine and scopolamine and our re-extraction
of certain samples, our method was validated and confirmed by its ability to extract and
analyse these alkaloids from solution or plant material. The recovery rate for standard
extraction was higher than 78% for atropine and 95% for scopolamine, while the efficacy of
the extraction was higher than 83% for atropine and 87% for scopolamine; the samples had
a relative standard deviation of 2.7%.
On average, the content of scopolamine in our samples was 692 µg/g (=0.07%),
while the content of atropine was approximately double at 1379 µg/g (=0.14%). This higher
content of hyoscyamine/atropine than scopolamine is consistent with research on other
closely related plants [11–16]. That being said, the content of these alkaloids changes
drastically between growth phases in related plants and may do so in S. carniolica as well,
though hyoscyamine/atropine is generally predominant [17].
As displayed in Table 1, the content of both alkaloids was greatly varied between
the different organs. However, the differences even within the organ groups are large; as
a result, the ANOVA test for the differences of scopolamine between the organs is only
marginally significant (p = 0.059). The differences for atropine, however, are significant
(ANOVA p = 0.006). Previous research in Slovenia has likewise shown a great deal of
variation between S. carniolica plants in their content of atropine and scopolamine [18],
a trend that was also observed in closely related plants with the same alkaloids [10].
Table 1. The content of scopolamine and atropine (µg/g) in different organs of S. carniolica (summarised for all locations, varieties, and years).
Scopolamine
Atropine
Total alkaloids
(Scopolamine +
atropine)
Flower
Leaf
Root
Total
Mean
619
1103
346
692
N
28
31
31
90
SD
748
1964
379
1273
Mean
492
1615
1945
1379
N
28
31
31
90
SD
460
2737
1328
1887
Mean
1110
2717
2291
2070
N
28
31
31
90
SD
1004
4621
1585
2967
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Our research exhibited the highest content of scopolamine in the leaves and lowest in
the roots, while the content of hyoscyamine/atropine was highest in the roots and lowest in
the flowers. The previously mentioned prior research in Slovenia completely matched this
pattern, except for finding that the highest quantity of scopolamine was in the flowers [18].
As S. carniolica and its close relatives are believed to synthesise hyoscyamine/atropine in
their roots, its high concentration there is unsurprising; similarly, as the flowers are the
most ephemeral part of the plant and most removed from the roots, it is reasonable that
this alkaloid would be least present there [10]. Hyoscyamine/atropine is also the precursor
for scopolamine, a change that is catalysed by hyoscyamine-hydroxylase (H6H); the higher
content of scopolamine in the leaves rather than the roots may suggest that this process
is largely carried out after the alkaloids leave the roots [10,19–21]. Further research with
higher significance values for the scopolamine differences is needed to confirm this.
The average content of each alkaloid was highest from the Bohor mountain collection
site (see Table 2), but only the differences for atropine were statistically significant (ANOVA
p = 0.017). The differences in scopolamine and the total alkaloid content were not significant
(p = 0.689 and p = 0.099 respectively). Seeing such a variation between sites was in line
with our predictions, as previous research showed a range of environmental factors such as
soil nitrogen, water stress, and pest type and number can affect the levels of these alkaloids
found in plants [22–25].
Table 2. The content of scopolamine and atropine (µg/g) in different locations of S. carniolica
(summarised for all organs, varieties, and years). The ANOVA p is 0.689 for scopolamine and
0.017 for atropine.
Bohor
Mountain
Ljubljana
Botanical
Garden
Pekel Pri
Borovnici
Mean
816
585
570
N
28
31
31
Scopolamine
SD
1695
794
381
Mean
1942
758
1445
N
28
31
31
SD
2428
965
1204
Atropine
We found no significant differences in the alkaloid content between different varieties/types of Scopolia carniolica (see Table 3). ANOVA p values were 0.800, 0.399, and 0.844
for scopolamine, atropine, and total alkaloids, respectively. Previous research with related
plants showed demonstrably different alkaloidal levels between varieties grown in the
same conditions, so this is either not a trait seen in S. carniolica, or the effect is concealed by
other (random or environmental) effects, or the two varieties are not distinct [26,27].
Table 3. The content of scopolamine and atropine (µg/g) in different varieties/types of S. carniolica
(summarised for all organs, locations, and years). The ANOVA p is 0.800 for scopolamine and 0.399
for atropine.
Mean
Scopolamine
Atropine
Hladnikiana
Hybrid?
Normal
Striped
Flowers
617
638
906
386
N
28
31
31
3
SD
1748
388
863
50
Mean
1313
1934
987
1363
N
28
31
31
3
SD
2446
1269
1137
1313
Plants 2021, 10, 1643
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Indeed, not only was the alkaloid difference not significant, but the reported morphological differences that are so clearly laid out in the literature did not present themselves at
all. The supposedly clear-cut distinction between the yellow-green flowers of the Hladnik
version and the purple outside and yellow inside of the main variety was a nebulous
continuum between the two [28]. Even referring to it as a continuum, however, is generous;
though many flowers were intermediary in their colours, we also collected an individual
with flowers in stripes of purple and yellow running the length of the corolla.
As previously stated, several other differences were noted with lesser frequency by
various authors, such as the Hladnik variety having shorter leaves, larger and fewer flowers,
and a later flowering time. These patterns were not observed in our collections, and in fact
the opposite was equally as likely to be true, making it seem that these were all within the
normal variability of the species. Similarly, the presence of purple veins was meant to be
present in only one variety but was observed in plants with a range of flower colours.
We found no significant differences in the alkaloid content between different years
of collection (see Table 4). Though many environmental conditions can shift and affect
alkaloid production within a site on a given year, it is clear that the two subsequent years
sampled for this research were similar enough as to not significantly impact this process.
Table 4. The content of scopolamine and atropine (µg/g) in S. carniolica collected in different years
(summarized for all organs, locations, and varieties). The ANOVA p is 0.799 for scopolamine, 0.724 for
atropine, and 0.908 for total alkaloids.
Scopolamine
Atropine
2019
2020
Mean
659
728
N
28
31
SD
1613
730
Mean
1446
1304
N
28
31
SD
2357
1163
To further analyse the data, we created four general linear models (Table 5), two
for each alkaloid. In one model we assumed that independent variables (organ, location,
type, and year) influence the dependant variables (scopolamine and atropine content)
without interaction. In the other model, we assumed there could be all possible two-way
interactions. The results were similar to our ANOVA analysis: atropine content is highly
influenced by organ type, which displays a weaker yet present influence on scopolamine
content. Location also proved to influence alkaloid content. The year of collection and
the variety of the plant, however, did not significantly impact the quantity of alkaloids
in the plants.
Plants 2021, 10, 1643
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Table 5. The results of general linear model.
Scopolamine
Source of
variability
df
Corrected model
23
Intercept
1
organ
2
Location
Type
Sig.
Sig.
Sig.
0.391
0.201
0.167
0.014
0.000
0.007
0.000
0.000
0.744
0.054
0.095
0.006
2
0.134
0.124
0.083
0.051
3
0.235
0.211
0.981
0.985
year 1
Location
Location × type
0×
type
organ × location
4
organ × locaLocation × year
tion 1
1 0.629
0.629 0.949
0.949 0.541
0.541 0.375
organ × type Location 6×
year
Type × year
2
organ × type
organ × year Type × year
2
Error
66
organ × year
Error
Total
90
Total
Corrected total
89
Corrected total
1 0.683
0.373
6
2 0.328
2
66
90
Plants 2021, 10, x FOR PEER REVIEW
year
Sig.
Atropine
0
4
na
0.452
0.130
na
0.452
0.130
0.683
0.373
0.328
na
0.380
0.459
0.657
0.367
0.503
5 of 9
0.375
na
0.380
0.459
0.657
0.367
0.503
89
As exhibited in Figure 1, the content of one alkaloid correlated with the content of the
Asall
exhibited
Figure
1, the
contentand
of one
alkaloid
with
content of
other (p = 0.000001 for
samplesinand
0.071,
0.000001
0.000317
forcorrelated
flower, leaf
andthe
root
respectively). the other (p = 0.000001 for all samples and 0.071, 0.000001 and 0.000317 for flower, leaf and
root respectively).
Figure 1. Grouped scatter plot of scopolamine and atropine by organ.
Figure 1. Grouped scatter plot of scopolamine and atropine by organ.
We performed a PCA analysis, where six dependent variables (atropine or scopola-
We performed
a PCA analysis, where six dependent variables (atropine or scopolamine
mine by leaf, root and flower) in individual specimens were used. The results of the PCA
by leaf, root and(see
flower)
specimens
were
used.
of the PCA
(see phenoFigurein2)individual
reinforce the
observation
about
the The
lack results
of classification
of visual
types by phytochemical variables.
Plants 2021, 10, 1643
6 of 9
Plants 2021, 10, x FOR PEER REVIEW
Figure 2) reinforce the observation about the lack of classification of visual phenotypes
6 of by
9
phytochemical variables.
Figure
Figure2.2.PCA
PCAanalysis
analysiswith
withsix
sixdependent
dependentvariables
variables(atropine
(atropineororscopolamine
scopolaminebybyleaf,
leaf,root
rootand
and
flower) in individual specimens.
flower) in individual specimens.
3.3.Materials
Materialsand
andMethods
Methods
3.1.Sample
SampleCollection
Collection
3.1.
Sampleswere
werecollected
collectedduring
duringflowering,
flowering,with
witha acollection
collectionperiod
periodofof1–9
1–9April
April2019
2019
Samples
and
24
March–4
April
2020.
A
total
of
31
plants
were
sampled
(8
of
the
common
variety,
and 24 March–4 April 2020. A total of 31 plants were sampled (8 of the common variety,
Hladnik’svariety,
variety,and
and88that
thatmorphologically
morphologicallyfell
fellbetween
betweenthe
thetwo,
two,all
allclassified
classifiedby
by
1515Hladnik’s
corolla
colour)
from
three
different
locations:
the
Ljubljana
botanical
garden
(eight
plants
corolla colour) from three different
Ljubljana botanical garden (eight plantsin
mountain
in2019
2019and
andsix
sixplants
plantsinin2020),
2020),Pekel
Pekelpri
priBorovnici
Borovnici(three
(threeplants
plantsinin2019),
2019),and
andBohor
Bohor
moun(six(six
plants
in 2019
andand
seven
plants
in 2020).
NotNot
all of
plants
hadhad
flowers.
tain
plants
in 2019
seven
plants
in 2020).
allthe
of the
plants
flowers.
3.2.Sample
SamplePreparation
Preparation
3.2.
Plants were divided into roots, flowers (including the pedicels), and leaves/stems.
Plants were divided into roots, flowers (including the pedicels), and leaves/stems.
Most had all three organ types available, but this was not always the case as some did not
Most had all three organ types available, but this was not always the case as some did not
have enough flowers for analysis; as a result, we had a total of 87 samples rather than 93.
have enough flowers for analysis; as a result, we had a total of 87 samples rather than 93.
Samples were then dried at room temperature and ground.
Samples were then dried at room temperature and ground.
3.3. Extraction
3.3. Extraction
The methods used for extraction and analysis were based upon previously published
The methods
foralterations.
extraction and analysis were based upon previously published
research
[29] withused
minor
research
[29]
with
minor
alterations.
A total of 0.25 g of the dried plant matter was weighed out and then transferred into
A
total
of 0.25byg rinsing
of the dried
was weighed
out andwas
thenthen
transferred
a 50 mL
beaker
withplant
3 × 5matter
mL methanol.
The beaker
covered into
with
a parafilm
50 mL beaker
by
rinsing
with
3
×
5
mL
methanol.
The
beaker
was
then
covered
with
into which small holes were poked and then put into an ultrasonic bath without
parafilm
intoheat
which
additional
for small
1 h. holes were poked and then put into an ultrasonic bath without
additional
heat
for
1
h. then filtered into a 50 mL round bottom flask using round format
The extract was
The
extract
was
then paper
filtered
into
50 mL(Macherey-Nagel,
round bottom flask
using
round format
cellulose qualitative filter
MN
615aGrade
Duren,
Germany).
A total
cellulose qualitative filter paper MN 615 Grade (Macherey-Nagel, Duren, Germany). A
total of 5 mL of methanol was used to rinse the beaker and filter into the flask afterwards.
The ethanolic extract was then completely evaporated in a rotary evaporator with a 40 °C
water bath at 200 mbar.
Plants 2021, 10, 1643
7 of 9
of 5 mL of methanol was used to rinse the beaker and filter into the flask afterwards.
The ethanolic extract was then completely evaporated in a rotary evaporator with a 40 ◦ C
water bath at 200 mbar.
The remaining dry extract was resuspended with 25 mL of 0.5% HCl and then poured
into a 250 mL separatory funnel. A total of 20 mL of dichloromethane was then used to
rinse the flask into the separatory funnel as well. The funnel was then capped and shaken
to carry out liquid-liquid extraction. The mixture was allowed to sit until separation of
the phases had occurred, and the lower layer (dichloromethane) was then removed and
discarded. Two more rounds of 20 mL dichloromethane were repeated in this manner.
Then 1 mL of 30–33% ammonium hydroxide solution was added to the remaining
(water) phase to raise the pH to 10. The sample was then shaken to ensure proper mixing.
A total of 20 mL of dichloromethane were added and extracted as previously, with the
only difference being that the dichloromethane was collected and kept. This process
was repeated with an additional four rounds of 20 mL of dichloromethane, collecting all
dichloromethane extracts into the same 250 mL beaker. The remainder (water phase) in the
separatory funnel was discarded after completion of this step.
Then 15 g of anhydrous sodium sulphate was added to the dichloromethane extract
and mixed. This was then filtered into a 100 mL round bottom flask, with an additional 2
mL of dichloromethane being used to rinse the beaker and filter.
This solution was evaporated in a rotary evaporator with a 40 ◦ C water bath at
700 mbar. Once evaporated, 2 × 2.5 mL of methanol were used to dissolve the dry extract
and transfer it into a test tube. For samples from which there was too little to attain
0.25 g of dried plant matter at the beginning of extraction (primarily the flower samples),
a corresponding amount of methanol was used in this step to ensure equal concentration
of the alkaloids.
Standards of atropine (Sigma, St. Louis, MO, USA) and scopolamine hydrochloride
(Fluka, Charlotte, NC, USA) were also extracted using the same extraction process to
ensure viability of the method; sample re-extraction was also performed to determine
effectiveness.
Samples were then filtered for HPLC and additional volume was stored in case of
further need.
3.4. Analysis
For identification via reference standards and quantification of the compounds, the
HPLC system (Shimadzu Prominence) was used. It consisted of a system controller
(CBM20A), a column oven (CPO-20AC), and a solvent delivery pump with a degasser
(DGU20A5) connected to a photodiode array detector (SPD-M20A) that monitored wavelengths 190–800 nm. The responses of the detectors were recorded using LabSolution
software version 5.71. The chromatography was performed at 40 ◦ C and a flow rate of 2 mL
min, using a Phenomenex Kinetex® C18 column (10 cm × 4.6 mm I.D., 2.7 µm particle size).
We utilised the following gradient method with solvent A (water with 2% acetonitrile and
0.1% trifluoroacetic acid) and solvent B (acetonitrile with 2% water and 0.1% trifluoroacetic
acid): 0–1 min 3% B, 1–10 min 3–20% B, 10–12 min 20–100% B and 12–15 min 100–5% B.
Samples and standards were detected at an absorbance of 210 nm and were injected into
the HPLC system at a volume of 10 µL.
4. Conclusions
The debate on the status of Scopolia carniolica var. Hladnikiana as a separate variety
from the main species is old, and unlikely to be settled simply through the present research.
In combining our results with established literature to compare, it seems more likely that
these morphological differences are a simple variation in the species rather than being
attributable to varietal labels.
As the first published chemical comparison of these supposed varieties, we observed
that there is in fact no significant connection between the morphological features and the
Plants 2021, 10, 1643
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content of its main alkaloids, hyoscyamine/atropine and scopolamine. Though this in itself
does not prove that these plants should all be considered without a varietal divide, it does
seem suggestive of such conclusions. The botanical observations noted during collection
further point in this direction; however, molecular work is needed on this topic to confirm
the level of difference between these organisms.
At this point it is worth noting that the most consistent variation between the two
supposed types was seen at the Ljubljana Botanical Garden site; indeed, this is where
the type collection for S. carniolica var. Hladnikiana that was found by Franc Hladnik was
planted and still grows. Perhaps Hladnik’s individual samples are indeed representative of
a distinct variety (perhaps his detractors would even go so far as to suggest he selectively
bred the plant in private to amplify its features before announcing it publicly) or perhaps
it is just another individual within the normal distribution of the species upon which a
varietal type was arbitrarily constructed. Future research using quantitative measurements
to examine the sizing of various morphological traits and studies able to quantify the colour
of the flowers is needed to reach further conclusions; similarly, future research should
focus on the chemical composition of these plants in terms of other substances besides the
alkaloids here investigated, to see if the chemical fingerprint of this plant holds any secrets
to its taxonomic troubles.
Author Contributions: Conceptualization, K.F.; methodology, S.K. and M.R.; investigation, K.F. and
M.R.; data curation, K.F.; formal analysis, S.K.; writing—original draft preparation, K.F.; writing—review
and editing, K.F., S.K. and M.R.; supervision, S.K.; funding acquisition, K.F. and S.K. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by University of Ljubljana Biotechnical Faculty’s financial
support fund for doctoral student research projects.
Data Availability Statement: All data is reported within the manuscript.
Acknowledgments: Botanični Vrt Univerze v Ljubljani for allowing collection from their gardens,
where the original specimen of S. carniolica var. hladnikiana discovered and collected by Franc Hladnik
still grows.
Conflicts of Interest: The authors declare no conflict of interest.
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