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Acta Sci. Pol. Hortorum Cultus, 17(4) 2018, 137–155 ISSN 1644-0692 e-ISSN 2545-1405 DOI: 10.24326/asphc.2018.4.13
ORIGINAL PAPER Accepted: 29.01.2018
STRUCTURE OF LEAVES AND PHENOLIC ACIDS
IN Kalanchoë daigremontiana Raym.-Hamet & H. Perrier
Mykhaylo Chernetskyy1, Anna Woźniak2, Agnieszka Skalska-Kamińska3, Beata Żuraw4 , Eliza Blicharska3, Robert Rejdak2, Helena Donica5, Elżbieta Weryszko-Chmielewska4
1 Botanical Garden, Maria Curie-Skłodowska University, Sławinkowska 3, 20-810 Lublin, Poland 2 Department of General Ophthalmology, Medical University of Lublin, Chmielna 1, 20-079 Lublin, Poland 3 Department of Analytical Chemistry, Medical University of Lublin, Chodźki 4a, 20-093 Lublin, Poland 4 Department of Botany, University of Life Sciences in Lublin, Akademicka 15, 20-950 Lublin, Poland 5 Department of Clinical Biochemistry, Medical University of Lublin, Staszica 16, 20-081 Lublin, Poland
ABSTRACT
Kalanchoë daigremontiana leaves contain phenolic compounds, which are one of the determinants of plant
therapeutic properties. Light and scanning electron microscopes were used to analyse the structure of
leaves. The main aims of the study included the analysis of the anatomy of leaves, localisation of phenolic
compounds, and identification of phenolic acids. The thickness of the amphistomatic leaf blades, the num-
ber, the size of stomata, and the value of stomatal index, as well as the structure of the parenchyma cells
have indicated that K. daigremontiana is adapted to arid environments. The histochemical assays revealed
the presence of phenolic idioblasts in the leaf blades and petioles. The idioblasts were located in the epi-
dermis, subepidermal layer, a deeper portion of the mesophyll, and in the sheaths of vascular bundles. The
phytochemical analyses of leaves demonstrated the presence of gallic, ferulic, caffeic, p-coumaric, and pro-
tocatechuic acids in the form of esters. We carried out the research of the anatomical structure of K. dai-
gremontiana leaves, which has been insufficiently documented to date. We have also revealed new localisa-
tion of phenolic compounds in the leaf tissues of this species.
Key words: Kalanchoë, tissues, phenolic idioblasts, phenolic acids
INTRODUCTION
There are over 150 species in the Kalanchoë
Adans. genus (Crassulaceae DC.) [Descoings
2006]. Kalanchoë daigremontiana Raym.-Hamet
et H. Perrier (syn. Bryophyllum daigremontianum
(Raym.-Hamet et H. Perrier) A. Berger) represents
the group Bulbilliferae Boiteau et Mannoni, sec-
tion Bryophyllum (Salisb.) Boiteau et Mannoni.
It originates from dry and hot semi-desert areas of
the south-west of Madagascar [Boiteau and Allor-
ge-Boiteau 1995]. The species was naturalised in
some tropical countries, e.g. in India [Descoings
2003]. K. daigremontiana is an ornamental pot
plant commonly cultivated in many countries
[Sarwa 2001].
Kalanchoë daigremontiana is a leaf succulent
with a life cycle dependent on environmental condi-
tions. It is usually a biennial and sometimes peren-
nial plant. It produces an unbranched, up to 1.5 m
© Copyright by Wydawnictwo Uniwersytetu Przyrodniczego w Lublinie
Chernetskyy, M., Woźniak, A., Skalska-Kamińska, A., Żuraw, B., Blicharska, E., Rejdak, R., Donica, H., Weryszko-Chmielewska, E. (2018). Structure of leaves and phenolic acids in Kalanchoë daigremontiana Raym.-Hamet & H. Perrier. Acta Sci. Pol. Hortorum Cultus, 17(4), 137–155. DOI: 10.24326/asphc.2018.4.13
www.hortorumcultus.actapol.net 138
high stem. The 20-cm leaves, dark green on the
adaxial side, are tapered at the apex. The lamina
margins are regularly serrated and exhibit purple
colouration on the abaxial side. K. daigremontiana
is characterised by vivipary. The spaces between the
teeth bear numerous adventitious buds or propagules
[Descoings 2003]. Propagules, i.e. the somatic em-
bryos of K. daigremontiana, are a transitional form
between adventitious buds and embryos [Batygina
et al. 1996]. Pendulous flowers with a red or purple
bell-shaped corolla form cymose panicles [Des-
coings 2003].
K. daigremontiana represents plants in which
photoperiodic factors exert an effect on the for-
mation of propagules. Upon an increase in the day
length to over 13 hours, K. daigremontiana plants
become viviparous [Chovanskaâ 1970, Kopcewicz
and Lewak 2005].
Many species representing the genus Kalanchoë
have long been used in traditional medicine [Costa
et al. 1995]. High pharmacological activity of
K. pinnata, i.e. an antibacterial, antiparasitic, anti-
cancer, anti-inflammatory, and cardioactive effect,
has been demonstrated [Joseph et al. 2011, Pattewar
2012]. Formulations from K. daigremontiana sap
exhibit anti-inflammatory properties as well.
The leaves of this species are used in ethnomedicine
to treat skin diseases and to staunch bleeding
[Sarwa 2001]. K. daigremontiana contains bufadi-
enolides, which reduce the proteolytic activity of
thrombin [Costa et al. 1995, Kołodziejczyk-Czepas
et al. 2017].
As other species from this genus, K. daigremon-
tiana has considerable antioxidant activity related to
the content of phenolic acids [Bogucka-Kocka et al.
2016]. Phenolic acids are an important group of sub-
stances with a variety of pharmacological anti-
inflammatory and fungistatic activities [Nowak and
Rychlińska 2012]. Many methods have been reported
in analyses of phenolic compounds: HPLC, capillary
electrophoresis, voltammetric [Tyszczuk et al. 2011],
gas chromatography. Planar chromatography was
also used [Wójcik-Kosior 2006]. The antioxidant
properties are also assigned to vitamin E present in
this species, whose novel form containing tocomo-
noenols was detected by Kruk et al. [2011]. Addi-
tionally, K. daigremontiana leaf tissues contain ster-
ols [Costa et al. 1995] and triterpenoids [Maarseveen
and Jetter 2009].
Previous anatomical investigations of Kalanchoë
species conducted by other authors demonstrated
phenolic idioblasts located in various leaf zones.
In K. crenata, K. daigremontiana, K. gastonis-
bonnieri, K laciniata, K. pinnata, and K. pumila,
phenolic idioblasts formed a regular or irregular layer
below epidermis and were evenly spaced in meso-
phyll [Balsamo and Uribe 1988, Chernetskyy and
Weryszko-Chmielewska 2008, Legramandi 2011,
Moreira et al. 2012, Brzezicka et al. 2015]. In the
leaves of K. crenata, K. gastonis-bonnieri, K. lacini-
ata, K. pinnata, and K. pumila, the phenolic idio-
blasts were irregularly distributed around vascular
bundles and hydathodes [Chernetskyy and Weryszko-
Chmielewska 2008, Leal-Costa et al. 2010,
Legramandi 2011, Moreira et al. 2012, Brzezicka et
al. 2015]. Phenolic compounds located in epidermal
cells near stomata were found only in K. crenata
[Moreira et al. 2012].
Since phenolic compounds are an important group
of biologically active compounds contained in Kal-
anchoë plant organs and their localisation and the
anatomy of K. daigremontiana leaves have not been
sufficiently explored, the aim of the study was: (i) to
show the leaf anatomy, (ii) to localise phenolic com-
pounds in tissues, and (iii) to perform qualitative
analysis of phenolic acids in fresh K. daigremontiana
leaves.
MATERIALS AND METHODS
Plant material The material for the anatomical study consisted of
typical K. daigremontiana leaves sampled from the
middle part of the stems of five biennial plants grow-
ing in the collection of greenhouse plants of the Bo-
tanical Garden, Maria Curie-Skłodowska University
in Lublin. The plants grew in plastic pots in a collec-
tion of succulents located in a well-lit greenhouse.
The cultivation conditions were characterised by an
average annual air temperature of 23°C and 64%
Chernetskyy, M., Woźniak, A., Skalska-Kamińska, A., Żuraw, B., Blicharska, E., Rejdak, R., Donica, H., Weryszko-Chmielewska, E. (2018). Structure of leaves and phenolic acids in Kalanchoë daigremontiana Raym.-Hamet & H. Perrier. Acta Sci. Pol. Hortorum Cultus, 17(4), 137–155. DOI: 10.24326/asphc.2018.4.13
www.hortorumcultus.actapol.net 139
humidity. In summer, the temperature during the day
reached 39°C. Leaves for anatomical analyses were
collected in June and July.
For phytochemical analysis, the leaves of a two-
year old K. daigremontiana plant were collected in July
from the garden of the Faculty of Pharmacy, Medical
University in Lublin. Six fully developed leaves were
taken from one plant for the investigations.
Anatomical analysis The morphological characteristics of the leaf
blade margins, adventitious buds, and propagules
were observed under a stereoscopic microscope
Olympus SZX 12.
The anatomical/histological examination of leaf
tissues was carried out with the use of a Nikon SE
light microscope with a scale in the ocular. Semi-
solid glycerol sections were made from fresh and
fixed in 70% ethanol leaves. Hand-made cross-
sections of the leaf and paradermal sections from the
upper part of fresh leaves were prepared. The follow-
ing morphological features were analysed: the densi-
ty of epidermal cells and stomata (per 1 mm2), the
location and length of stomata. The following ana-
tomical characteristics of leaves were analysed: the
leaf blade thickness in its middle part on the midrib
and between the midrib and the margin, the height of
epidermal cells and the thickness of their outer walls,
the mesophyll structure and diameter of parenchyma
cells, the location of phenolic idioblasts, and the dis-
tribution of vascular bundles.
For morphometric analyses, two mature leaves
were collected from each of the five examined plants.
Next, semi-fixed preparations were made and
3 measurements for each trait were performed. The
measurements were performed for 30 stomata.
To visualise lignified cell walls, some leaf cross
sections were treated with 5% phloroglucine
(ca. 1 min.) and 15% hydrochloric acid (HCl, rinsed
in redistilled water and sealed in a 70% glycerol solu-
tion. Potassium dichromate [Gabe 1968] or toluidine
blue [Ramalingam and Ravindranath 1970] were
used as histochemical assays for localisation of phe-
nolic compounds in the leaf tissues. Phenolic com-
pounds stained orange or dark brown when treated
with potassium dichromate, whereas green or blue
colour was obtained after toluidine blue treatment.
Based on the number of epidermal cells and sto-
mata per leaf unit area, the stomatal index (I) was
calculated; it expresses the percent ratio of stomata to
the number of epidermal cells with stomata in the
analysed area:
S × 100a
I = S + E
(%)
S – number of stomata per 1 mm2,
E – number of epidermal cells per 1 mm2. The analysis of the leaf surface micromorphology
was carried out using a BS 300 Tesla scanning elec-
tron microscope (SEM). Fragments of the leaf blade
between the midrib and margin were collected from
5 leaves. Leaf fragments were fixed in 2% glutaral-
dehyde with 2.5% paraformaldehyde in 0.075 M
phosphate buffer at pH 6.8 and 4°C for 12 h. Next,
they were washed twice in the buffer each time for
15 minutes and in redistilled water for the same
time. Subsequently, the samples were dehydrated in
the increasing acetone series (30, 50, 70, 90, and
100%) for 30 minutes in each change. After dehy-
dration, the material was critical point dried in liq-
uid CO2 and sputtered with gold using a CS 100
Sputter Coater.
The preparations were assessed for the morpholo-
gy and location of stomata, the shape of anticlinal
walls and the surface of the outer walls of epidermis,
the presence of wax structures, and the sculpture of
the cuticle surface.
Qualitative phytochemical analysis by thin-layer chromatography – densitometric method
Phenolic acid standards of the highest grade,
listed in Table 1, were purchased from Sigma (St.
Louis, MO, USA) and prepared as 0.1% solutions in
methanol.
Caffeic acid for the spectrophotometric assay was
prepared as a stock solution at a concentration of
0.1% of methanol. The range of standard concentra-
tions from 0.005 to 0.08% was prepared to construct
a calibration graph.
Chernetskyy, M., Woźniak, A., Skalska-Kamińska, A., Żuraw, B., Blicharska, E., Rejdak, R., Donica, H., Weryszko-Chmielewska, E. (2018). Structure of leaves and phenolic acids in Kalanchoë daigremontiana Raym.-Hamet & H. Perrier. Acta Sci. Pol. Hortorum Cultus, 17(4), 137–155. DOI: 10.24326/asphc.2018.4.13
www.hortorumcultus.actapol.net 140
Table 1. Investigated compounds
No. Common name Phenolic acid
1 p-Coumaric acid 4-Hydroxycinnamic acid
2 Gallic acid 3,4,5-Trihydroxybenzoic acid
3 Vanillic acid 4-Hydroxy-3-methoxybenzoic acid
4 Chlorogenic acid ester of chinic and caffeic acid
5 Caffeic acid 3,4-Dihydroxycinnamic acid
6 Salicylic acid 2-Hydroxybenzoic acid
7 Syringic acid 3,5-Dimethoxy-4-hydroxybenzoic acid
8 Protocatechuic acid 3,4-Dihydroxybenzoic acid
9 Ferulic acid 4-Hydroxy-3-methoxycinnamic acid
A Exammined extract
Table 2. Composition of mobile phases in phenolic acid planar chromatography analysis
Kind
of stationary
phase
Composition of mobile phase
Development program
Number of
development cycles
Distance of development
(mm)
Si 60 tertbuthyl-methyl ether + hexane + acetic acid +
metanol; 3.5 : 5.0 : 0.5 : 0.2 (v/v) 2 85
Si 60 DIOL
tertbuthyl-methyl ether + heptane + formic acid +
metanol + toluene; 3.0 : 4.0 : 0.5 : 0.3 : 2.0 (v/v) 3 85
tertbuthyl-methyl ether + heptane + formic acid +
toluene; 3.0 : 4.0 : 2.0 : 2.0 (v/v) 1 85
tertbuthyl-methyl ether + heptane + formic acid +
metanol + toluene; 3.0 : 4.0 : 2.0 : 0.5 : 2.0 (v/v) 3 20
All solvents used in the experiments and for sam-
ple preparation purchased from Polish Reagents
(POCh, Gliwice, Poland) were pro analysis grade.
In the present publication, high performance thin lay-
er chromatography with densitometry was applied for
qualitative analysis of phenolic acids in a K. daigremon-
tiana methanol extract of fresh leaves of the plant.
The method for extraction and purification of
phenolic acids was based on literature data with some
modifications [Tyszczuk et al. 2011]. For sample
preparation, triple exhaustive extraction (15 min
each) in an ultrasonic bath (at 55°C) (Sonorex Type
RK 102 HB Bandelin, Berlin, Germany) was con-
ducted in three replicates. 10 g of fresh, crushed plant
leaves and three portions (50 mL) of the extractant
(methanol) were taken each time.
The extracts were pooled and evaporated to
dryness in a rotary evaporator (HB Basic RV 05-ST,
IKA, Łódź, Poland) under reduced pressure.
The dry residue was washed with portions of hot
distilled water (about 30 mL in total) at 4°C for 24 h.
Then the solution was filtrated with the use of a paper
filter and defatted by shaking out twice in a separator
with petroleum ether (10 mL each time). Subsequent-
ly, the water extract solution was extracted ten times
with portions (10 mL) of diethyl ether in the separa-
tor. The ether extracts were combined and shaken ten
times with a 5% water solution of NaHCO3 (10 mL
each time) to receive water-soluble phenolic acid
salts. The extract was next acidified up to pH 3 (with
36% HCl) and extracted ten times with 10 mL por-
tions of diethyl ether in the separator. The ether solu-
tion of free phenolic acids was dried with the use of
Na2SO4 anhydrate, filtrated, and evaporated. The dry
residue was dissolved in 5 mL of methanol and used
in chromatographic experiments.
Chernetskyy, M., Woźniak, A., Skalska-Kamińska, A., Żuraw, B., Blicharska, E., Rejdak, R., Donica, H., Weryszko-Chmielewska, E. (2018). Structure of leaves and phenolic acids in Kalanchoë daigremontiana Raym.-Hamet & H. Perrier. Acta Sci. Pol. Hortorum Cultus, 17(4), 137–155. DOI: 10.24326/asphc.2018.4.13
www.hortorumcultus.actapol.net 141
Fig. 1. Densitogram of setting the analytical lambda for chosen phenolic acids
Chernetskyy, M., Woźniak, A., Skalska-Kamińska, A., Żuraw, B., Blicharska, E., Rejdak, R., Donica, H., Weryszko-Chmielewska, E. (2018). Structure of leaves and phenolic acids in Kalanchoë daigremontiana Raym.-Hamet & H. Perrier. Acta Sci. Pol. Hortorum Cultus, 17(4), 137–155. DOI: 10.24326/asphc.2018.4.13
www.hortorumcultus.actapol.net 142
Chromatography was performed on 10 cm ×
10 cm HPTLC F254 plates coated with silica gel and
silica gel modified with DIOL groups (Merck, Darm-
stadt, Germany). First, the most common [Sowa et al.
2013] silica gel with high performance quality was
used. The multiply gradient development (MGD)
technique with an experimentally chosen composition
of mobile phases (tab. 2) was applied. The plates
were washed with methanol and acetone and dried in
a stream of hot air before use. Samples (8 μL) and
standards (3 μL) were applied to the plates as 5 mm
bands by means of a Hamilton syringe. Chromato-
grams were developed in horizontal Teflon DS
chambers (Chromdes, Lublin, Poland). The develop-
ment programs are presented in Table 2. The spots
were detected and preliminarily identified by UV
illumination at λ = 245 nm and λ = 366 nm and doc-
umented with the use of a digital camera. The
HPTLC-Diol plates were also derivatized with the
use of a reagent specific to phenolic compounds:
a mixture of sulfanilic acid with a 5% solution of
sodium nitrate in equal parts, which gives phenolic
acids yellow to brown colour of spots.
The presence of phenolic acids in the extract of
K. daigremontiana was confirmed with the use of
densitometric measurements. Silica gel plates were
scanned for each identified compound to set the ana-
lytical lambda (fig. 1). By multivalve scan of stand-
ards’ spots, the analytical lambda with maximum ab-
sorbance was set (fig. 1). After chromatogram devel-
opment, the plates were subjected to densitometric
scanning with the use a Desaga CD 60 densitometer
(Desaga, Heidelberg, Germany) controlled with
a Pentium computer, in which the peaks of the stand-
ards of the investigated phenolic acids corresponded
with the ones from the extract (figs 6 A, 6 B).
Then, the extract and the standard were scanned in
the established conditions. The chromatogram on
DIOL plates with the best separation parameters was
densitometrically scanned at 254 nm.
Quantitative spectrometric analysis. An extract
of the plant with methanol was prepared for spectro-
photometric analysis. 10 g of fresh leaves were ex-
tracted three replicates, with 50 mL of the extractant
each time, in a boiling water bath EkoTerm TW12
(Julabo, Sellbach, Germany) under reflux. The com-
bined methanol extracts were filtrated with the use of
filter paper and evaporated to dryness in a rotary evap-
orator HB Basic RV 05-ST (IKA, Łódź, Poland) under
reduced pressure. The dry residue was dissolved in
20 mL of hot water and left in a refrigerator for
12 hours. After filtration, the water solution was dilut-
ed up to 100 mL in a volumetric flask.
The content of phenolic acids, calculated as caf-
feic acid, was determined using the spectroscopic
method with the Arnov’s reagent (sodium molybdate
(10 g) and sodium hydroxide (10 g) dissolved in
water in a 100 mL volumetric flask) described in The
Polish Pharmacopoeia IX [2011].
Five different volumes of the stock solution were
used for preparation of the calibration graph. 5 mL of
distilled water, 1 mL of a hydrochloric acid solution
(0.5 mol L–1), 1 mL of Arnov’s reagent and, after
6 min., 1 mL of sodium hydroxide solution (1 mol L–1)
were also added to five 10 mL volumetric flasks.
The flasks were filled up with distilled water to the
mark. Measurements of absorbance at 490 nm
(Spectrophotometer Genesis, Thermo Scientific,
Chicago, USA) were done for each concentration
exactly after 3 minutes from the last reagent addi-
tion in a glass cell of 1 cm. A mixture of all rea-
gents without the caffeic acid standard was used as
a reference. Measurements of the extract were per-
formed in the same conditions (glass cell, reference
sample composition). Six samples of the investigat-
ed extract were added instead of the caffeic acid
standards to the reagent mixture and measured at
λ = 490 nm.
RESULTS
Leaf anatomy. The fleshy K. daigremontiana
leaves are characterised by a cylindrical petiole
(fig. 2 A, B) and a bifacial, peltate leaf blade
(fig. 2 A, C) with a mean thickness of 3820.0 μm in
the midrib and 1913.3 μm between the margin and
midrib (tab. 3). The leaf blade margin and the abaxial
side of the leaf are purple (fig. 2 B–F). The apex of
the teeth in the lamina margin bears hydathodes
(fig. 2 D, F), and adventitious buds (fig. 2 D, E), from
which propagules are formed (fig. 2 C, D), develop
between the teeth on lingulate appendages.
Chernetskyy, M., Woźniak, A., Skalska-Kamińska, A., Żuraw, B., Blicharska, E., Rejdak, R., Donica, H., Weryszko-Chmielewska, E. (2018). Structure of leaves and phenolic acids in Kalanchoë daigremontiana Raym.-Hamet & H. Perrier. Acta Sci. Pol. Hortorum Cultus, 17(4), 137–155. DOI: 10.24326/asphc.2018.4.13
www.hortorumcultus.actapol.net 143
Fig. 2. Upper part of a Kalanchoë daigremontiana plant and fragments of leaves with propagules: A – plant apex with
fleshy leaves and numerous propagules at the leaf blade margins; B – base of the peltate part of the leaf blade in lateral
view; C – apical portion of the leaf blade with propagules (asterisks) at the margins; D – fragment of the leaf blade margin
with a propagule (asterisk); ligulate appendages with adventitious buds (arrows) visible between the teeth; the presence of
epithem hydathodes at the apex of the teeth (arrowheads); E – ligulate appendage with an adventitious bud (arrow) at the
leaf blade margin; F – hydathode at the apex of the lamina margin tooth. Scale bars: A, E, F = 1 mm, B–D = 5 mm
K. daigremontiana leaves are covered by single-
layered epidermis (figs 3 A, B, 4 C). The epidermal
cells on the adaxial side of the leaves are approx.
1.3-fold higher than the cells of the abaxial epidermis
(tab. 3). In the top view, they have irregular isodia-
metric or slightly elongated shapes (fig. 3 A, B). On
the abaxial surface, they are nearly 2-fold smaller
than on the adaxial surface, as evidenced by their
number per unit area (tab. 3). The cells of the petiole
epidermis have a prosenchymatic shape (fig. 3 C).
The anticlinal walls of the epidermal cells are usually
undulating in their outlines (figs 3 A, B). The outer
walls of these cells are thickened and covered by
a cuticle (fig. 4 C) providing the leaves with a shiny
coating (fig. 2 A, C). The surface of the outer walls is
slightly convex (figs 3A, D, 4 C) and the cuticle
is smooth (fig. 3 A) or undulating (fig. 3 A, D).
The cuticle bears striated ornamentation only on
subsidiary cells (fig. 3 A). Wax patches are present
on the cuticle surface (fig. 3 A, D).
K. daigremontiana leaves are amphistomatic.
There are many anisocytic stomatal complexes in
the epidermis of both lamina surfaces (fig. 3 A, B),
with a 2-fold higher number on the abaxial than
adaxial side (tab. 3). Subsidiary cells surrounding
the stomata have a different shape and are smaller
than the other epidermal cells (fig. 3 A–C). The
length of the leaf blade stomata on the adaxial and
Chernetskyy, M., Woźniak, A., Skalska-Kamińska, A., Żuraw, B., Blicharska, E., Rejdak, R., Donica, H., Weryszko-Chmielewska, E. (2018). Structure of leaves and phenolic acids in Kalanchoë daigremontiana Raym.-Hamet & H. Perrier. Acta Sci. Pol. Hortorum Cultus, 17(4), 137–155. DOI: 10.24326/asphc.2018.4.13
www.hortorumcultus.actapol.net 144
abaxial surfaces is in the range of 28–30 µm
(tab. 3). The stomata are located at the level of the
other epidermal cells. The stomatal cuticle forms
thickened, convex outer cuticular ledges (fig. 3 A,
D). Based on the number of stomata and epidermal
cells (per 1 mm2 of the leaf blade area), the stomatal
index calculated for both surfaces of the K. daigre-
montiana leaves differs only by 1% (tab. 3). Some
stomatal complexes are present in the leaf petiole
epidermis as well (fig. 3 C).
Fig. 3. Surface of Kalanchoë daigremontiana leaf epidermis – A, C, D (SEM), B (LM): A – fragment of the adaxial leaf
blade surface with stomata (arrows), wax patches (double arrows) and thickened anticlinal walls of epidermal cells; B –
fragment of the epidermis on the abaxial surface of the leaf blade with stomata (arrows) and undulating anticlinal cell
walls; C – elongated epidermal cells with a stoma (arrow) on the leaf petiole surface; D – stomata (arrows) and wax patch-
es (double arrows) on the adaxial leaf blade epidermis. Scale bars: A–D = 20 µm
Chernetskyy, M., Woźniak, A., Skalska-Kamińska, A., Żuraw, B., Blicharska, E., Rejdak, R., Donica, H., Weryszko-Chmielewska, E. (2018). Structure of leaves and phenolic acids in Kalanchoë daigremontiana Raym.-Hamet & H. Perrier. Acta Sci. Pol. Hortorum Cultus, 17(4), 137–155. DOI: 10.24326/asphc.2018.4.13
www.hortorumcultus.actapol.net 145
Table 3. Mean measurement results and some structural traits of Kalanchoë daigremontiana leaf blade (n = 30)
Leaf blade thickness
In the midrib (μm) 3820.0 ±13.47
Between the margin and the midrib (μm) 1913.3 ±10.79
Epidermis traits
Height of cells (μm) ad 34.10 ±1.70
ab 26.70 ±1.13
Number of cells per 1 mm2 area ad 227.2 ±1.64
ab 425.3 ±1.89
Length of stomata (μm) ad 30.04 ±0.53
ab 28.42 ±0.73
Number of stomata per 1 mm2 area ad 21.2 ±0.84
ab 45.3 ±1.09
Stomatal index (%) ad 8.54
ab 9.62
Diameter of mesophyll cells
Subepidermal mesophyll (μm) ad 78.3 ±15.47
ab 39.7 ±13.05
Middle part of the mesophyll (μm) max. 160.0 ±19.32
min. 123.6 ±17.89 ad – adaxial surface of the leaf blade
ab – abaxial surface of the leaf blade
max. – maximal diameter
min. – minimal diameter
The chlorenchymatic tissue in the K. daigremon-
tiana leaves is not differentiated into the palisade and
spongy parts; it forms a small-celled mesophyll under
the epidermis and a large-celled water-bearing paren-
chyma in the middle part of the leaf (fig. 4 A, B). The
subepidermal mesophyll in the leaf blades usually
consists of one or several layers of small, closely
adherent cells (fig. 4 B, C).
The cells of the subepidermal mesophyll on the
adaxial side of the leaf blade are generally 2-fold larger
than such cells on the abaxial side. In the cross section
of the leaf blades, the diameter of the cells of these mes-
ophyll layers is approx. 39.7 µm (abaxial layer) and
78.3 µm (adaxial layer) (tab. 3). They are 2–4-fold
smaller than the cells of the water-bearing chlorenchy-
ma in the middle part of the leaf. The cells of the water-
storing chlorenchyma in the middle part of the leaf have
diameters of 160.0 × 123.6 µm (tab. 3); their shape
factor is in the range of 1.2–2.0, with a mean value of
1.3 and determines the ellipsoidal shape of the cells of
this tissue.
The central vascular bundles in the leaves are sur-
rounded by perivascular sheaths composed of tiny
mesophyll cells (fig. 4 D).
Between the epidermis and mesophyll in the
K. daigremontiana leaf petioles, there are 1–3 layers
of compact angular collenchyma (fig. 4 C). The
number of collenchyma layers in the petiole clearly
decreases from its base to the lamina. The subepi-
dermal angular collenchyma is fragmentarily distrib-
uted on the abaxial side of the leaf blade below the
vascular bundle. The size of the collenchyma cells is
similar to that of the small mesophyll cells.
The sclerenchyma in the leaves is located around
larger vascular bundles in the form of fibres constituting
a mestome sheath. It is the best-developed tissue in the
leaf petioles and near the main vascular bundle in the
leaf blades (fig. 4 D). The vascular bundles represent the
closed collateral type. In the central veins in the petiole
and the lamina veins located on their extension are visi-
ble three large bundles (fig. 4 A). The cross sections
show fine lateral vascular bundles surrounding large
bundles in the petioles and leaf blades (fig. 4 A, B).
Chernetskyy, M., Woźniak, A., Skalska-Kamińska, A., Żuraw, B., Blicharska, E., Rejdak, R., Donica, H., Weryszko-Chmielewska, E. (2018). Structure of leaves and phenolic acids in Kalanchoë daigremontiana Raym.-Hamet & H. Perrier. Acta Sci. Pol. Hortorum Cultus, 17(4), 137–155. DOI: 10.24326/asphc.2018.4.13
www.hortorumcultus.actapol.net 146
Fig. 4. Tissues of Kalanchoë daigremontiana leaves in cross sections (LM): A – cross section of the leaf petiole base with
visible vascular bundles (VB); B – fragment of a cross section of the leaf blade with visible vascular bundles (VB);
C – fragment of a cross section of the petiole; layers of angular collenchyma cells (Co) present under a single-layered
epidermis (E) with thickened outer cell walls; D – cross section of the main vascular bundle of the leaf blade with visible
xylem (X), phloem (Ph), and sclerenchyma (Sc); E, F – cross section of a fragment of leaf blade epidermis treated with
toluidine blue. Visible content of phenolic compounds in vacuoles; G–J – phenolic idioblasts (ID) dispersed in the meso-
phyll (G, H) and surrounding vascular bundles (I, J); fresh leaves treated with potassium dichromate. Scale bars:
A, B = 1 mm, C, D, G–J = 50 µm, E, F = 10 µm
Chernetskyy, M., Woźniak, A., Skalska-Kamińska, A., Żuraw, B., Blicharska, E., Rejdak, R., Donica, H., Weryszko-Chmielewska, E. (2018). Structure of leaves and phenolic acids in Kalanchoë daigremontiana Raym.-Hamet & H. Perrier. Acta Sci. Pol. Hortorum Cultus, 17(4), 137–155. DOI: 10.24326/asphc.2018.4.13
www.hortorumcultus.actapol.net 147
Fig. 5. Phenolic idioblasts (ID) visible in Kalanchoë daigremontiana petiole and lamina tissues treated with tolui-
dine blue (LM, fresh leaves): A – fragment of the main vascular bundle from the petiole cross sections with
a visible xylem (X), phloem (Ph), and phenolic idioblast (ID); B–D – phenolic idioblasts (ID) form a partial (B) or
complete (C) envelope around the bundles or 1–3 layers at the bundles (D). Scale bars: A–D = 50 µm
The different tissues of the K. daigremontiana
leaf contain numerous phenolic idioblasts accumulat-
ing phenolic compounds in their vacuoles. The local-
isation of phenolic idioblasts was confirmed with the
use of the histochemical assays. Phenolic compounds
are present in epidermal cells (fig. 4 E, F) and in the
subepidermal layer. Phenolic idioblasts are dispersed
in the parenchyma as single cells or form multicellu-
lar aggregates (fig. 4 G, H). They are also located in
close proximity to the vascular elements (fig. 4 I, J).
There are few phenolic idioblasts around the large
vascular bundles in the leaf petioles (fig. 5 A). They
surround the smaller vascular bundles partially (fig. 5 B)
or completely (fig. 5 C). 1–3 layers of these cells are
visible near some bundles in the leaf petioles (fig. 5 D).
Identification of phenolic acids. For preliminary
identification of phenolic acids, comparison of reten-
tion factor (RF) values with RF of standard substances
was conducted. A combination of two stationary
phases was applied for separation of the extract com-
ponents. In the described conditions, three phenolic
acids: protocatechuic, p-coumaric, and ferulic in the
K. daigremontiana extract were separated and identi-
fied (figs 6 A, 6 B). Simultaneously, the presence of
salicylic and vanillic acid was not confirmed. Given
the doubts about chlorogenic, caffeic, and syringic
acid, sequential experiments were carried out. Satis-
factory separation of two phenolic acids, caffeic and
gallic, was achieved on silica gel modified with
DIOL groups (fig. 7). Additionally, the presence of
Chernetskyy, M., Woźniak, A., Skalska-Kamińska, A., Żuraw, B., Blicharska, E., Rejdak, R., Donica, H., Weryszko-Chmielewska, E. (2018). Structure of leaves and phenolic acids in Kalanchoë daigremontiana Raym.-Hamet & H. Perrier. Acta Sci. Pol. Hortorum Cultus, 17(4), 137–155. DOI: 10.24326/asphc.2018.4.13
www.hortorumcultus.actapol.net 148
Fig. 6 A. Densitometric identification of phenolic acids in the methanol extract of fresh
Kalanchoë daigremontiana leaves
Chernetskyy, M., Woźniak, A., Skalska-Kamińska, A., Żuraw, B., Blicharska, E., Rejdak, R., Donica, H., Weryszko-Chmielewska, E. (2018). Structure of leaves and phenolic acids in Kalanchoë daigremontiana Raym.-Hamet & H. Perrier. Acta Sci. Pol. Hortorum Cultus, 17(4), 137–155. DOI: 10.24326/asphc.2018.4.13
www.hortorumcultus.actapol.net 149
Fig. 6 B. Densitometric identification of phenolic acids in the methanol extract
of fresh Kalanchoë daigremontiana leaves
chlorogenic acid, which was supposed to be detected
on silica gel plates, was eliminated. No content of
vanillic, chlorogenic, salicylic, and syringic acid from
the group of free phenolic acids was confirmed in the
K. daigremontiana methanol extract.
Visualisation of phenolic acids. For better identi-
fication of phenolic acids on DIOL plates, the method
of plate derivatization with sulfuric acid and sodium
nitrate was used. Visualisation of spots under light of
320 nm and sulfuric acid derivatization confirmed the
presence of gallic acid in the extract. Consequently,
the spots of caffeic and protocatechuic acids had the
same values and colour. Identification of caffeic acid
was conducted in UV light at λ = 254 nm in plates
without the derivatization reagent. The colour of the
spot was light blue.
With the use of two stationary phases and a com-
bination of three visualization techniques, five phe-
nolic acids in the methanol extract of fresh leaves of
K. daigremontiana were separated and identified.
The presence of ferulic, gallic, caffeic, p-cou-
maric, and protocatechuic acid in the extract was
confirmed. Selected chromatograms are presented in
Figs 7 and 8.
Chernetskyy, M., Woźniak, A., Skalska-Kamińska, A., Żuraw, B., Blicharska, E., Rejdak, R., Donica, H., Weryszko-Chmielewska, E. (2018). Structure of leaves and phenolic acids in Kalanchoë daigremontiana Raym.-Hamet & H. Perrier. Acta Sci. Pol. Hortorum Cultus, 17(4), 137–155. DOI: 10.24326/asphc.2018.4.13
www.hortorumcultus.actapol.net 150
Fig. 7. Chromatogram of phenolic acids on a DIOL-silica plate in UV light
at λ = 254 nm. Numbers refer to the phenolic acid names shown in Table 1
Fig. 8. Chromatogram of phenolic acids on a silica plate in UV light
at λ = 254 nm after derivatization with sulfuric acid and sodium ni-
trate. Numbers refer to the phenolic acid names shown in Table 1
Chernetskyy, M., Woźniak, A., Skalska-Kamińska, A., Żuraw, B., Blicharska, E., Rejdak, R., Donica, H., Weryszko-Chmielewska, E. (2018). Structure of leaves and phenolic acids in Kalanchoë daigremontiana Raym.-Hamet & H. Perrier. Acta Sci. Pol. Hortorum Cultus, 17(4), 137–155. DOI: 10.24326/asphc.2018.4.13
www.hortorumcultus.actapol.net 151
Spectroscopic analysis. The spectroscopic meth-
od with the Arnov’s reagent was used for quantita-
tive determination of free phenolic acids from
K. daigremontiana. In the spectroscopic analysis,
the calibration graph of the dependence between the
absorbance and concentration of caffeic acid was
drafted. The total content of phenolic acids is
2 µg·10 g–1 in fresh K. daigremontiana leaves.
DISCUSSION
Leaf structure. Stomatal complexes in repre-
sentatives of the genus Kalanchoë belong to the
anisocytic type characterised by the presence of
three subsidiary cells surrounding the stomata
[Metcalfe and Chalk 1957]. There are usually
greater numbers of stomata on the abaxial than
adaxial side of leaves [Sharma and Lewis 1987],
which was also shown in this study. The abaxial
side of the K. daigremontiana leaves had smaller
stomata than those located on the adaxial side. As
reported by Sharma and Lewis [1987], the number
of stomata and undulation of the anticlinal walls of
epidermal cells in representatives of the genus Kal-
anchoë has a diagnostic importance.
Other authors reported that the number of stomata
per unit area in Kalanchoë blossfeldiana, K. daigre-
montiana, and K. tubiflora leaves reached up to
50 mm–2, likewise in other succulents and is several
times lower than that observed in mesophytes
[Strobel and Sunberg 1983–1984. A similar range of
the number of stomata was demonstrated in the
K. daigremontiana analysed in the present study and
in previously investigated species from this genus
[Chernetskyy 2006, Chernetskyy and Weryszko-
Chmielewska 2008]. The number of stomata per
1 mm2 area in K. orgyalis and K. tomentosa ranged
from 54 to 92 depending on the side of the leaf
[Chernetskyy 2006]. The author of the latter study
suggests that the density of stomata, small sizes of
epidermal cells, presence of trichomes, and reduced
sizes of stomata in the leaves of these species may be
a result of advanced xeromorphism associated with
adaptation to arid environments. In K. daigremon-
tiana, besides fully mature stomata, underdeveloped
stomata in different stages of development were ob-
served, which may limit transpiration in adverse con-
ditions.
Sharma and Dunn [1968] reported that a greater
number of stomata and greater cuticle thickening
with granular texture and waxy coating were ob-
served in K. fedtschenkoi leaves in dry and hot condi-
tions than in wet habitats. The number and structure
of cells surrounding the stomata were unchanged,
regardless of the conditions. The authors found that
the stomatal index was not a stable diagnostic feature
for this taxon, as it changes in different ecological
conditions. The highest stomatal index was calculated
for control plants and specimens growing in desert
conditions, while the lowest value was reported for
plants growing in wet conditions.
The assimilation tissue in K. daigremontiana
leaves is not differentiated into palisade and sponge
mesophyll but is divided into a small-celled subepi-
dermal mesophyll and a large-celled water-bearing
CAM-type mesophyll in the middle part of the leaf.
Similar mesophyll differentiation was reported in
previous studies of this and other species from the
genus Kalanchoë [Balsamo and Uribe 1988, Cher-
netskyy and Weryszko-Chmielewska 2008, Abdel-
Raouf 2012, Brzezicka et al. 2015]. In the anatomi-
cal characteristics of the family Crassulaceae,
Metcalfe and Chalk [1957] report that palisade tis-
sue is rarely present in the leaves of the taxa of this
family.
In the species analysed in this study, the cells of
the large-celled mesophyll contain chloroplasts and
are involved in the photosynthesis process as well as
water storage. The main adaptation trait in leaf succu-
lents is accumulation of water in the parenchyma of
deeper leaf layers, which is associated with the pres-
ence of large vacuoles in the cells. In highly special-
ised leaf succulents (e.g. Aloë, Haworthia, Lithops,
Salsola, and some Peperomia,), the central part of
leaves is composed of a specialised water-bearing
tissue, whose large cells only occasionally contain
chloroplasts [Troll 1959, Kaul 1977, Chauser-
-Volfson et al. 2002, Nuzhyna and Gaydarzhy 2015].
Similarly to other genera from the family Crassu-
laceae, this tissue in genus Kalanchoё representatives
Chernetskyy, M., Woźniak, A., Skalska-Kamińska, A., Żuraw, B., Blicharska, E., Rejdak, R., Donica, H., Weryszko-Chmielewska, E. (2018). Structure of leaves and phenolic acids in Kalanchoë daigremontiana Raym.-Hamet & H. Perrier. Acta Sci. Pol. Hortorum Cultus, 17(4), 137–155. DOI: 10.24326/asphc.2018.4.13
www.hortorumcultus.actapol.net 152
is replaced by water-bearing mesophyll with smaller
cells containing many chloroplasts [Lyubimov et
al. 1986].
The present study indicates that, in the K. daigre-
montiana leaves, cells containing phenolic com-
pounds were present in the epidermal and subepider-
mal tissue, forming a continuous or discontinuous
layer. They were also located singly near the vascular
elements or surrounded the entire bundles as
1–3 layers. We also found phenolic idioblasts dis-
persed singly or in groups in the mesophyll. In turn,
Balsamo and Uribe [1988] reported that phenolic
idioblasts in K. daigremontiana leaves were located
only in the subepidermal layer and were evenly
spaced throughout the mesophyll. Our research has
provided new data concerning the location of phenol-
ic idioblasts in this species, as we found them in the
epidermis and around the vascular bundles, where
they even formed multi-layer bundle sheath. The
presence of tannin-containing cells was detected in
leaves of other species from the genus Kalanchoë
[Chernetskyy and Weryszko-Chmielewska 2008,
Legramandi 2011, Chernetskyy 2012, Brzezicka et al.
2015]. The content of phenolic compounds was also
observed in some cells of the stalks of non-glandular
trichomes in K. millotii [Weryszko-Chmielewska and
Chernetskyy 2005].
Chernetskyy and Weryszko-Chmielewska [2008]
found well visible chloroplasts in the cytoplasm of
K. pumila phenolic idioblasts, which were several
fold smaller than chloroplasts contained in mesophyll
cells. Similarly, there were chloroplasts in
K. daigremontiana cells accumulating phenolic com-
pounds. Single starch grains and spherical osmophilic
structures were noted in K. pumila cells. The elec-
tron-dense central vacuoles of such cells exhibited
dark flocculent content. Such features of the structure
of phenolic idioblasts have been shown in other plant
species as well [Bačić et al. 2004].
The content of phenolic compounds in plant cells
is an important adaptive trait, as the compounds play
a protective role by absorption of UV radiation,
which is unfavourable to plant organs, and protect
plants from pathogenic agents and damage caused by
entomofauna [Oleszek et al. 2001, Kopcewicz and
Lewak 2005].
Phenolic compounds Some of Kalanchoë species are supposed to have
many positive effects on the human organism [Schol-
tysik et al. 1986, Muzitano et al. 2006, Kamboj and
Saluja 2009]. The main group of illnesses treated by
Kalanchoë originates from oxidative-stress. The ef-
fectiveness of plants in healing the diseases comes
from the high amount of phenolic derivatives. The
presence of phenolic compounds determines the anti-
inflammatory, wound healing, and free radical scav-
enging activity of the plant [Sazhina et al. 2014].
Although K. daigremontiana is well known for medi-
cal applications, its exact chemical composition is
documented very poorly. The concentration of phe-
nolic acids in a free form in a plant was described by
Bogucka-Kocka et al. [2016] as 124 µg·g–1 of dry-
weight. The authors of the cited study found the highest
content of ferulic, protocatechuic, and caffeic acids in
the analysed K. daigremontiana material. Fresh
leaves were subjected to two kinds of extraction:
accelerated solvent extraction and maceration with
ethanol. In the studies reported by these authors,
quantification of phenolic acids with the HPLC
method was conducted after extract concentration
and filtration.
The presented publication describes the qualita-
tive content of phenolic acids in a hydrolysed metha-
nol extract. Phenolic acids described in our publica-
tion occur in the plant in the form of esters. Exhaus-
tive extraction with sonification was used. A combi-
nation of two stationary phases and gradient elution
was used in the HPTLC method.
The content of phenolic acids presented in this
study is partially similar to previous investigations
conducted by other authors. We confirmed the pres-
ence of gallic, ferulic, caffeic, p-coumaric, and proto-
catechuic acids. We did not detect chlorogenic and
syringic acids, which were found with the HPLC
method by Bogucka-Kocka et al. [2016]. We also
investigated salicylic and vanilic acid but their pres-
ence was not confirmed.
The presented quality investigations are the first
step in the analysis of phenolic acids from K. dai-
gremontiana. So far, we have determined the sum of
phenolic acids from esters with organic acids.
The knowledge of the content of basic compounds in
Chernetskyy, M., Woźniak, A., Skalska-Kamińska, A., Żuraw, B., Blicharska, E., Rejdak, R., Donica, H., Weryszko-Chmielewska, E. (2018). Structure of leaves and phenolic acids in Kalanchoë daigremontiana Raym.-Hamet & H. Perrier. Acta Sci. Pol. Hortorum Cultus, 17(4), 137–155. DOI: 10.24326/asphc.2018.4.13
www.hortorumcultus.actapol.net 153
K. diagemontiana with confirmed medical activity is
still very poor; therefore, future investigations are
planned e.g. quantification analysis of particular phe-
nolic acids and hydrolysis of glycosides.
CONCLUSIONS
1. We have demonstrated in the study that the
fleshy Kalanchoë daigremontiana leaves are charac-
terised by bifacial, amphistomatic leaf blades with
the abaxial epidermis bearing an approximately
2-fold greater number of stomatal complexes than the
adaxial surface. The leaf parenchyma is differentiated
into a small-celled subepidermal mesophyll and
a large-celled mesophyll located in the central part of
the leaf. Both mesophyll types contain chloroplasts.
2. The results of the histochemical assays, indicate
the content of phenolic compounds in various lamina
and petiole tissues, i.e. in the cells of the epider-
mis, subepidermal and perivascular parenchyma
(1–3 layers), and in the cells scattered in the leaf
mesophyll, which is partly new information about
this species. The phenolic compounds in the different
leaf tissues are contained at substantial levels, which
are probably reflected in the therapeutic activity.
3. The phytochemical analyses have evidenced the
presence of gallic, ferulic, caffeic, p-coumaric, and
protocatechuic acids representing phenolic com-
pounds in the ester form in Kalanchoë daigremontia-
na leaves.
ACKNOWLEDGEMENTS
Research supported by Poland’s Ministry of Sci-
ence and Higher Education as part of the statutory
activities of the Department of Botany, University of
Life Sciences in Lublin.
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