STRUCTURE OF LEAVES AND PHENOLIC ACIDS alanchoë ... · Planar chromatography was also used...

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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

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© 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%



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.



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


toluene; 3.0 : 4.0 : 2.0 : 2.0 (v/v) 1 85


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.



Fig. 1. Densitogram of setting the analytical lambda for chosen phenolic acids



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.



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



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



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).



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



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



Fig. 6 A. Densitometric identification of phenolic acids in the methanol extract of fresh

Kalanchoë daigremontiana leaves



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.



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



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



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



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.

REFERENCES

Abdel-Raouf, H.S. (2012). Anatomical traits of some spe-

cies of Kalanchoë (Crassulaceae) and their taxonomic

value. Ann. Agric. Scien., 57(1), 73–79. DOI:

10.1016/j.aoas.2012.03.002.

Bačić, T., Ljubešc, N., Užarević, Z., Grgić, L., Roša, J.

(2004). TEM investigation of tannins and chloroplast

structure in needles of damaged silver fir trees (Abies

alba Mill.). Acta Biol. Cracov. Ser. Bot., 46, 145–149.

Balsamo, R., Uribe, E. (1988). Leaf anatomy and ultra-

structure of the Crassulacean-acid-metabolism plant

Kalanchoë daigremontiana. Planta, 173(2), 183–189.

DOI: 10.1007/BF00403009.

Batygina, T.B., Bragina, E.A., Titova, G.E. (1996). Mor-

phogenesis of propagules in viviparous species Bryo-

phyllum daigremontianum and B. calycinum.

Acta Soc. Bot. Pol. 65(1-2), 127–133. DOI:

10.5586/asbp.1996.022.

Bogucka-Kocka, A., Zidon, Ch., Kasprzycka, M.,

Szymczak, G., Szewczyk, K. (2016). Phenolic acid

content, antioxidant and cytotoxic activieties of four

Kalanchoë species. Saudi J. Biol. Sci. DOI:

10.1016/j.sjbs.2016.01.037 [in press].

Boiteau, P., Allorge-Boiteau, L. (1995). Kalanchoë de

Madagascar. Systématique, écophysiologie et phyto-

chemie. Karthala, Paris.

Brzezicka, E., Karwowska, K., Kozieradzka-Kiszkurno,

M., Chernetskyy, M. (2015). Leaf micromorphology of

Kalanchoë laciniata (Crassulaceae). Mod. Phytomor-

phol., 8, 49–52. DOI: 10.5281/zenodo.159829.

Chauser-Volfson, E., Shen, Z., Hu, Z., Gutterman, Y.

(2002). Anatomical structure and distribution of sec-

ondary metabolites as a peripheral defence strategy in

Aloë hereroensis leaves. Bot. J. Linn. Soc., 138, 107–

116. DOI: 10.1046/j.1095-8339.2002.00012.x.

Chernetskyy (Czernecki), M. (2006). Mikromorfologia

epidermy liści wybranych gatunków Kalanchoë Adans.

Rocznik EKPiUU (Lublin), 3, 371–380 [in Polish].

Chernetskyy, M. (2012). The role of morpho-anatomical

traits of the leaves in the taxonomy of Kalanchoideae

Berg. subfamily (Crassulaceae DC.). Modern Phyt., 1,

15–18. DOI: 10.5281/zenodo.162711.

Chernetskyy, M., Weryszko-Chmielewska, E. (2008).

Structure of Kalanchoë pumila Bak. leaves (Crassu-

laceae DC.). Acta Agrobot., 61(2), 11–24. DOI:

10.5586/aa.2008.029.

Chovanskaâ, N.V. (1970). Charakter fotoperiodičeskoj

lokalizacii v vegetativnom razmnoženii Kalanchoë

daigremontiana. In: Sbornik trudov po agronomičeskoj

fizike. Vol. 21. Svetofiziologiâ rastenij, Moškov, B.S.

(ed.). Kolos, Leningrad, 67–70.

Costa, S.S. Jossang, A. Bodo, B. (1995). Propriétés bi-

ologiques et phytochimie des Kalanchoë. In: Kalan-

choë de Madagaskar. Systématique, écophysiologie et

phytochemie, Boiteau, P., Allorge-Boiteau, L. (eds).

Karthala, Paris, 219–235.



Descoings, B., (2003). Kalanchoë. In: Illustrated Hand-

book of Succulent Plants – Crassulaceae, Eggli, U.

(ed.), Springer-Verlag, Berlin–Heidelberg–New York,

143–181.

Descoings, B. (2006). Le genre Kalanchoë (Crassulaceae):

structure et définition. J. Bot. Bull. Soc. Bot. Fr., 33, 3–

28.

Gabe, M. (1968). Techniques Histologiques. Masson et

Cie, París.

Joseph, B., Sridhar, S., Sankarganesh, Justinraj, Edwin,

B.T. (2011). Rare medicinal plant – Kalanchoë pinna-

ta. Res. J. Microbiol., 6(4), 322–327. DOI:

10.3923/jm.2011.322.327.

Kamboj, A., Saluja, A.K. (2009). Bryophyllum pinnatum

(Lam.) Kurz.: Phytochemical and pharmacological pro-

file: A reviev. Pharmacogn. Rev., 3, 364–374.

Kaul, R.B. (1977). The role of the multiple epidermis in

foliar succulence of Peperomia (Piperaceae). Bot. Gaz.,

138(2), 213–218. DOI: 10.1086/336917.

Kołodziejczyk-Czepas, J., Sieradzka, M., Moniuszko-

Szajwaj, B., Pecio, Ł., Ponczek, M.B., Nowak, P., Sto-

chmal, A. (2017). Bufadienolides from Kalanchoë dai-

gremontiana as thrombin inhibitors – In vitro and in

silico study. Int. J. Biol. Macromol., 99, 141–150. DOI:

10.1016/j.ijbiomac.2017.02.051.

Kopcewicz, J., Lewak, S. (ed.). (2005). Fizjologia roślin.

Wyd. Nauk. PWN, Warszawa.

Kruk, J., Pisarski, A., Szymańska, R. (2011). Novel vita-

min E forms in leaves of Kalanchoë daigremontiana

and Phaseolus coccineus. J. Plant. Physiol., 168, 2021–

2027. DOI: 10.1016/j.jplph.2011.06.015.

Leal-Costa, M.W., Nascimento, L.B.S., Moreira, N.S.,

Reinert, F., Costa, S.S., Lage, C.L.S., Tavares, W.S.

(2010). Influence of blue light on the leaf morphoana-

tomy of in vitro Kalanchoë pinnata (Lamarck) Persoon

(Crassulaceae). Microsc. Microanal., 16, 576–582.

DOI: 10.1017/S1431927610000279.

Legramandi, V.H.P. (2011). Kalanchoë gastonis-bonnieri

Raym.-Hamet et H. Perrier e Kalanchoë pinnata Pers.

(Crassulaceae): atividade antifúngica e estudo farma-

cognóstico comparativo. Dissertação apresentada ao

Programa de Pós-Graduação em Ciências Farmacêuti-

cas, da Faculdade de Ciências Farmacêuticas, UNESP,

como parte dos requisitos para obtenção do Título de

Mestre em Ciências Farmacêuticas, Araraquara – SP.

Available: ttps://repositorio.unesp.br/bitstream/handle/

11449/96246/legramandi_vhp_me_arafcf.pdf?sequence

=1 [date of access: 16.01.2018].

Lyubimov, V.Yu., Atakhanov, B.O., Bil, K.Ya. (1986).

Adaptation of photosynthetic apparatus in plants of the

Kara-Kum desert to extreme conditions. Russ. J. Plant

Physiol., 33(5), 888–895.

Maarseveen, C., Jetter, R. (2009). Composition of the

epicuticular and intracuticular wax layers on Kalan-

choë daigremontiana (Hamlet et Perr. De la Bathie)

leaves. Phytochemistry, 70(7), 899–906. DOI:

10.1016/j.phytochem.2009.04.011.

Metcalfe, C.R., Chalk, L. (1957). Anatomy of the dicotyle-

dons. Vol. 2. Clarendon Press, Oxford.

Moreira N.S., Nascimento, L.B.S., Leal-Costa M.V.,

Tavares E.S. (2012). Comparative anatomy of leaves of

Kalanchoë pinnata and K. crenata in sun and shade

conditions, as a support for their edentification. Rev.

Bras. Farmacogn. Braz. J. Pharmacogn., 22(5), 929-

936. DOI: 10.1590/S0102-659X2012005000056.

Muzitano, M.F., Tinoco, L.W., Guette, C., Kaiser, C.R.,

Rossi-Bergmann, B., Costa, S.S. (2006). The an-

tileishmanial activity assessment of unusual flavonoids

from Kalanchoë pinnata. Phytochemistry, 67, 2071–

2077. DOI: 10.1016/j.phytochem.2006.06.027.

Nowak, S., Rychlińska, I. (2012). Phenolic acids in the

flowers and leaves of Grindelia robusta Nutt. and

Grindelia squarrosa Dun. (Asteraceae). Acta Pol.

Pharm., 69, 693–698.

Nuzhyna, N.V., Gaydarzhy, M.M. (2015). Comparative

characteristics of anatomical and morphological adap-

tations of plants of two subgenera Haworthia Duval to

arid environmental conditions. Acta Agrobot., 68(1),

23–31. DOI: 10.5586/aa.2015.006.

Oleszek, W., Głowniak, K., Leszczyński, B. (eds) (2001).

Biochemiczne oddziaływania środowiskowe. Akad.

Med., Lublin.

Pattewar, S.V. (2012). Kalanchoë pinnata: phytochemical

and pharmacological profile, IJPSR 3(4), 993–1000.

DOI: 10.7439/ijpp.v2i1.223.

Ramalingam, K., Ravindranath, M.H. (1970). Histo-

chemical significance of green metachromasia to to-

luidine blue. Histochemie, 24(4), 322–327. DOI:

10.1007/BF00278217.

Sarwa, A. (2001). Wielki leksykon roślin leczniczych.

Książka i Wiedza, Warszawa.

Sazhina, N.N., Lapshin, P.V., Zagoskina, N.V., Korot-

kova, E.I., Misin, V.M. (2014). A comparative anal-

ysis of the antioxidant activity of Kalanchoë juices.

Russ. J. Bioorg. Chem., 40(7), 771–776. DOI:

10.1134/S1068162014070152.



Scholtysik, G., Wagner, H., Fische, M., Rüegg, U. (1986).

Cardiac glycoside-like effects of a bufadienolide ex-

tracted from Kalanchoë daigremontiana. In: Cardiac

Glycosides, Erdmann, E., Greef, K., Skou, J.C. (eds).

Steinkopff, Heidelberg, 1785–1985.

Sharma, G.K, Dunn, D.B. (1968). Effect of environment

on the cuticular features in Kalanchoë fedtschenkoi.

Bull. Torrey Bot. Club, 95(5), 464–473. DOI:

10.2307/2483478.

Sharma, G.K., Lewis, P.B. (1987). Cuticular differentiation

in Kalanchoë fedtschenkoi and Kalanchoë daigremon-

tianum (Crassulaceae). J. Tenn. Acad. Sci., 62(4),

97–98.

Sowa, I., Kocjan, R., Wójciak-Kosior, M., Świeboda, R.,

Zajdel, D., Hajnos, M. (2013). Physicochemical

properities of silica gelcoated with a thin layer of poly-

aniline (PANI) and its application in non-suppressed

ion chromatography. Talanta, 115, 451–456. DOI:

10.1016/j.talanta.2013.05.071.

Strobel, D.W., Sunberg, M.D. (1983–1984). Stomatal

density in leaves of various xerophytes-preliminary

study. J. Minn. Acad. Sci., 49(2), 7–9.

The Polish Pharmacopoeia IX (2011). Urząd Rejestracji

Produktów Leczniczych, Wyrobów Medycznych i

Produktów Biobójczych (URPLWMiPB), Warsaw.

Troll, W. (1959). Allgemeine Botanik. Ferdinand Enke

Verlag, Stuttgart.

Tyszczuk, K., Skalska-Kamińska, A., Woźniak, A. (2011).

Voltammetric method using a lead film electrode

for the determination of caffeic acid in a plant mate-

rial. Food Chem., 125, 1498–1503. DOI:

10.1016/j.foodchem.2010.10.075.

Weryszko-Chmielewska, E., Chernetskyy, M. (2005).

Structure of trichomes from the surface of leaves of

some species of Kalanchoë Adans. Acta Biol. Cracov.

Ser. Bot., 47(2), 15–22.

Wójciak-Kosior, M., Skalska, A. (2006). Thin-layer chro-

matography of phenolic acids on aminopropylsilica.

J. Planar Chromatogr., 19, 200–203. DOI:

10.1556/JPC.19.2006.3.5.

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