Mangiferin and Hesperidin Transdermal Distribution and Permeability through the Skin from Solutions and Honeybush Extracts (Cyclopia sp.)—A Comparison Ex Vivo Study

Abstract

Polyphenolic compounds—mangiferin and hesperidin—are, among others, the most important secondary metabolites of African shrub Cyclopia sp. (honeybush). The aim of this study was to compare the percutaneous absorption of mangiferin and hesperidin from solutions (water, ethanol 50%, (v/v)) and extracts obtained from green and fermented honeybush (water, ethanol 50%, (v/v)). Research was performed with the Bronaugh cells, on human dorsal skin. The mangiferin and hesperidin distributions in skin layers (stratum corneum, epidermis, and dermis) and in acceptor fluid (in every 2, 4, 6, and 24 h) were evaluated by HPLC–Photodiode Array Coulometric and Coulometric Electrochemical Array Detection. The transdermal distribution of hesperidin was also demonstrated by fluorescence microscopy. Results indicated that mangiferin and hesperidin were able to cross the stratum corneum and penetrate into the epidermis and dermis. An advantage of hesperidin penetration into the skin from the water over ethanol solution was observed (451.02 ± 14.50 vs. 357.39 ± 4.51 ng/cm2), as well as in the mangiferin study (127.56 ± 9.49 vs. 97.23 ± 2.92 ng/cm2). Furthermore, mangiferin penetration was more evident from nonfermented honeybush ethanol extract (189.85 ± 4.11 ng/cm2) than from solutions. The permeation of mangiferin and hesperidin through the skin to the acceptor fluid was observed regardless of whether the solution or the honeybush extract was applied. The highest ability to permeate the skin was demonstrated for the water solution of hesperidin (250.92 ± 16.01 ng/cm2), while the hesperidin occurring in the extracts permeated in a very low capacity. Mangiferin from nonfermented honeybush ethanol extract had the highest ability to permeate to the acceptor fluid within 24 h (152.36 ± 8.57 ng/cm2).

Full text

molecules
Article
Mangiferin and Hesperidin Transdermal Distribution
and Permeability through the Skin from Solutions
and Honeybush Extracts (Cyclopia sp.)—A Comparison
Ex Vivo Study
Anna Hering 1, * , Jadwiga Renata Ochocka 1, *, Helena Baranska 2, Krzysztof Cal 2
and Justyna Stefanowicz-Hajduk 1, *


Citation: Hering, A.; Ochocka, J.R.;
Baranska, H.; Cal, K.;
Stefanowicz-Hajduk, J. Mangiferin
and Hesperidin Transdermal
Distribution and Permeability
through the Skin from Solutions and
Honeybush Extracts (Cyclopia sp.)—A
Comparison Ex Vivo Study. Molecules
2021,26, 6547. https://doi.org/
10.3390/molecules26216547
Academic Editor: Francesco Cacciola
Received: 12 October 2021
Accepted: 26 October 2021
Published: 29 October 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Department of Biology and Pharmaceutical Botany, Medical University of Gdansk, 80-416 Gdansk, Poland
2Department of Pharmaceutical Technology, Medical University of Gdansk, 80-416 Gdansk, Poland;
helena.baranska@gmail.com (H.B.); krzysztof.cal@gumed.edu.pl (K.C.)
*Correspondence: anna.hering@gumed.edu.pl (A.H.); jadwiga.ochocka@gumed.edu.pl (J.R.O.);
justynastef@gumed.edu.pl (J.S.-H.)
Abstract:
Polyphenolic compounds—mangiferin and hesperidin—are, among others, the most
important secondary metabolites of African shrub Cyclopia sp. (honeybush). The aim of this study
was to compare the percutaneous absorption of mangiferin and hesperidin from solutions (water,
ethanol 50%, (v/v)) and extracts obtained from green and fermented honeybush (water, ethanol 50%,
(v/v)). Research was performed with the Bronaugh cells, on human dorsal skin. The mangiferin and
hesperidin distributions in skin layers (stratum corneum, epidermis, and dermis) and in acceptor fluid
(in every 2, 4, 6, and 24 h) were evaluated by HPLC–Photodiode Array Coulometric and Coulometric
Electrochemical Array Detection. The transdermal distribution of hesperidin was also demonstrated
by fluorescence microscopy. Results indicated that mangiferin and hesperidin were able to cross
the stratum corneum and penetrate into the epidermis and dermis. An advantage of hesperidin
penetration into the skin from the water over ethanol solution was observed
(451.02 ±14.50
vs.
357.39
±
4.51 ng/cm
2
), as well as in the mangiferin study (127.56
±
9.49 vs. 97.23
±
2.92 ng/cm
2
).
Furthermore, mangiferin penetration was more evident from nonfermented honeybush ethanol
extract (189.85
±
4.11 ng/cm
2
) than from solutions. The permeation of mangiferin and hesperidin
through the skin to the acceptor fluid was observed regardless of whether the solution or the
honeybush extract was applied. The highest ability to permeate the skin was demonstrated for the
water solution of hesperidin (250.92
±
16.01 ng/cm
2
), while the hesperidin occurring in the extracts
permeated in a very low capacity. Mangiferin from nonfermented honeybush ethanol extract had the
highest ability to permeate to the acceptor fluid within 24 h (152.36 ±8.57 ng/cm2).
Keywords:
skin penetration; skin permeation; HPLC; Fabaceae; honeybush; fluorescent microscopy
1. Introduction
Natural products used in the form of cosmetics or nutraceuticals nowadays are gaining
importance. They are usually well known in ethnomedicine as a relatively cheap and safe
source of active compounds [
1
]. There are many synthetic products on the market that are
dermatologically effective, though they cause unwanted side-effects. The most commonly
observed are redness, irritation, rashes, and itching. In addition, such products cannot
be often used in the area of injured skin, on mucosa, or in high amounts, due to the toxic
systemic effects [
2
]. Exposure to biotic and abiotic stress generates oxidative stress among
skin macromolecules and, in consequence, their dysfunction or degradation [
3
–
5
]. Due
to the fact that skin is considered the most external and first barrier of the human body,
its arrangement is essential to maintain internal homeostasis. The first layer—the stratum
corneum, is composed of dead, keratin-filled cells. However, they contain substances
Molecules 2021,26, 6547. https://doi.org/10.3390/molecules26216547 https://www.mdpi.com/journal/molecules

Molecules 2021,26, 6547 2 of 16
with hygroscopic properties, responsible for binding water, and enzymes participating in
metabolic processes, the activity of which is responsible for the acidic pH of the skin surface.
The specific arrangement of the stratum corneum and the hydrophobic properties create
this impenetrable barrier layer to pathogens and foreign substances [
3
,
6
,
7
]. Penetration
through the stratum corneum barrier is the basic parameter in studies of the transdermal
distribution of xenobiotics [
8
,
9
]. With the growing interest in the percutaneous route of
administration of medicinal substances as an alternative to oral intake, intensive research
has been started on increasing the permeability of the stratum corneum layer [
7
]. The main
problems of those studies are: determining whether the introduced substance should target
the dermis or reach the bloodstream; physical and biological properties of a xenobiotic;
the drug partition between cumulation in the skin and permeation through the skin;
individual differences and skin diseases [
9
–
12
]. Considering polyphenols as almost perfect
antioxidants, which can be used to protect the skin and its macromolecules against oxidative
stress [
13
,
14
], their ability to penetrate into and permeate through skin layers should be
under consideration. The permeability depends, among others, on the structure of the
molecules and their chemical properties; however, the transdermal distribution of these
compounds could be modified by the composition of the delivery system [15,16].
Polyphenols are a group of pharmacologically active, secondary metabolites, widely
spread in nature. It is confirmed that polyphenols have ability to protect skin macro-
molecules from internal and external degradation. Natural sources of specific polyphenolic
compositions combined with high quantitative content are in demand [1].
Cyclopia spp. (Fabaceae) is an endemic shrub growing wild and cultivated on planta-
tions only within Cape Province in the Republic of South Africa. Leaves and branches from
honeybush are collected and dried (green honeybush) or fermented and dried (fermented
honeybush), to prepare the tea beverage with many health properties [
17
]. The richness in
polyphenolic compounds, low tannins, and lack of caffeine have made this sweet-tasting
tea beverage a highly attractive alternative for Camellia sinensis tea [
18
,
19
]. Additionally,
extracts from honeybush are traditionally used by the local people in different skin dis-
eases [
17
]. The tisane is becoming increasingly more popular in commercial use among
consumers, and researchers.
The pharmacological activity of Cyclopia spp. is caused by the synergic effect of
polyphenolic fraction—xanthones, flavanones, flavones, isoflavones, coumestans, pheno-
lic acids, and others [
20
]. Therefore, extracts from honeybush have strong antioxidant
properties with a large amount of mangiferin and hesperidin, compounds well known as
oxidative stress fighters [
17
,
20
–
26
]. These compounds, among others, are also responsible
for the skin UV protection [
20
,
27
] and inhibition of wrinkle formation [
28
]. In addition,
Bartoszewski et al. indicated that mangiferin and hesperidin are safe for keratinocytes [
29
].
Mangiferin, a C-glucosyl xanthone, is widely distributed in nature. It is presented
abundantly in the families Anacardiaceae [
30
], Liliaceae [
31
], or Fabaceae [
18
]. The many
important healing pharmacological activities of mangiferin include analgesic, immunomod-
ulatory, antibacterial, and antidiabetic [
32
–
34
]. One of the major properties of mangiferin is
the lowering level of oxidative stress, which promotes in the long-term many degradation
diseases [
35
–
37
]. Despite numerous
in vitro
analyses confirming mangiferin activity, its
bioavailability is limited by both low solubility and absorption from the intestine [
38
,
39
].
Among the new routes of improving mangiferin bioavailability is topical application. It was
confirmed that mangiferin has the ability to pass through the stratum corneum, penetrate
the epidermis and dermis, and inhibit the degradation of collagen and elastin, as well as
wrinkle formation [
40
,
41
]. Additional results have confirmed mangiferin’s ability to inter-
act with macromolecules of the skin and increase the regenerative capacity of wounded
skin [
42
]. Mangiferin has a high potential in dermatology; however, many aspects of the
compound action in the skin have not yet been investigated.
Hesperidin, a flavanone (hesperetin-7-O-rutinoside), is abundantly presented in cit-
rus fruits [
43
,
44
]. Mostly known from cardiovascular protection and anti-inflammatory
functions, hesperidin has proved significant systemic effects, which are limited by the low

Molecules 2021,26, 6547 3 of 16
water solubility and low absorption from the intestine [
45
–
51
]. In recent years, derma-
tological studies have focused on hesperidin’s ability to improve the skin functions and
condition [
52
]. The transdermal activity of hesperidin is mainly related to its antioxidant
properties. It has been confirmed that hesperidin catalyses the scarring process through
the reduction of oxidative stress around the wounded area [
52
,
53
], and enhances the ability
to protect keratinocytes against UV-induced damage [54–56]. It has also been proven that
hesperidin inhibits the inflammatory reaction generated by keratinocytes when exposed to
oxidative stress [
57
]. In addition, hesperidin, by reducing free radicals generated in the
course of the melanogenesis process, inhibits melanogenesis and melanosome transport,
resulting in skin lightening [
58
–
60
]. The topical application of hesperidin improved skin
barrier permeability [
61
]. What is important is that the compound has no toxic effects after
neither oral nor topical application, whereas many ingredients present in cosmetic products
may cause allergic reactions and be responsible for adverse systemic effects [
52
,
62
–
65
].
Until now, animal models or tissue cultures have been used in studies of hesperidin, while
the transdermal distribution of this polyphenol after application on human skin has never
been analysed.
In this study, we estimated the permeation and distribution of mangiferin and hes-
peridin among skin layers ex vivo after the application of two types of solutions (water and
ethanol) and Cyclopia sp. extracts (water and ethanol). The results of Cyclopia sp. extracts,
as well as hesperidin permeation and transdermal distribution, were shown in this work
for the first time.
2. Results
Extracts A, B, C, and D (Table 1), as well as water or ethanol solutions of hesperidin or
mangiferin, were applied on the skin surface. After 2, 4, 6, and 24 h, samples of acceptor
fluid were taken to quantitatively analyse both compounds. The estimation of hesperidin
and mangiferin distribution among skin layers was determined after 24 h of experiment,
followed by skin layers’ separations and extractions. For the quantitative determination of
mangiferin and hesperidin in water and ethanol extracts applied on the skin (summarised
in Table 1), and in solutions extracted from individual layers of the skin, as well as in
acceptor fluids, HPLC analysis with electrochemical detection was utilised.
Table 1.
Summary of content of mangiferin and hesperidin [ng/mL] in Cyclopia sp. extracts analysed
by HPLC-UV.
Cyclopia
sp.
Extract
Type of Plant Material Solvent for the Extracts
Preparation Mangiferin Hesperidin
Anonfermented (“green”) H2O 1593.1 ±31.08 483.6 ±16.74
Bfermented H2O 967.2 ±22.17 376.13 ±7.07
Cnonfermented (“green”) 50% EtOH (v/v) 4695.68 ±65.33 423.98 ±13.01
Dfermented 50% EtOH (v/v) 2962.05 ±62.14 304.5 ±9
The ex vivo experiments revealed that mangiferin [
40
] and hesperidin from solutions
(water or ethanol) or extracts (water or ethanol) are capable of passing through the stratum
corneum and penetrating into the deeper layers of the skin, as well as permeating through
the skin.
2.1. Hesperidin Permeation into the Skin from Solutions and Honeybush Extracts
The ability of hesperidin to cross the skin barrier—the stratum corneum—has been
estimated and demonstrated in Figure 1and Table 2.

Molecules 2021,26, 6547 4 of 16
Table 2.
Accumulation of hesperidin analysed by HPLC-UV and HPLC-EC among skin layers: stratum corneum, epidermis,
and dermis, and hesperidin permeation through the skin after 2, 4, 6, and 24 h (ng/cm
2
) from the application of hesperidin
solutions (HeH2O, HeEtOH) or honeybush extracts (A, B, C, D).
HeH2O HeEtOH Extract A Extract B Extract C Extract D
S.c. 77.74 ±5.44 56.77 ±12.59 59.03 ±0.84 40.21 ±9.9 64.82 ±17.39 48.87 ±8.68
Epidermis 147.9 ±8.35 115.03±0.5 66.56 ±0.9 69.54 ±8.51 68.92 ±11 88.85 ±16.68
Dermis 225.38 ±29.72 185.59 ±0.45 121.85 ±0.56 107.33 ±12.56 80.21 ±12.05 87.44 ±5.78
2 h 83.18 ±12.79 71.33 ±12.38 5.08 ±1.22 6.72 ±1.84 8.43 ±0.83 10.31 ±3.06
4 h 117.18 ±12.79 88.13 ±20.04 8.87 ±2.35 8 ±1.35 11.18 ±0.7 12 ±2.91
6 h 151.08 ±22.35 108.77 ±14.38 10.97 ±0.38 9.13 ±0.38 12.51 ±3.91 14.31 ±2.91
24 h 250.92 ±16.01 132 ±14.93 16.72 ±4.22 13.23 ±4.24 17.79 ±3.84 22.41 ±1.56
S.c. stratum corneum; each average value was obtained from three independent repetitions.
Molecules2021,26,xFORPEERREVIEW4of16


Table2.AccumulationofhesperidinanalysedbyHPLC‐UVandHPLC‐ECamongskinlayers:stratumcorneum,epider‐
mis,anddermis,andhesperidinpermeationthroughtheskinafter2,4,6,and24h(ng/cm2)fromtheapplicationofhes‐
peridinsolutions(HeH2O,HeEtOH)orhoneybushextracts(A,B,C,D).
HeH2OHeEtOHExtractAExtractBExtractCExtractD
S.c.77.74±5.4456.77±12.5959.03±0.8440.21±9.964.82±17.3948.87±8.68
Epidermis147.9±8.35115.03±0.566.56±0.969.54±8.5168.92±1188.85±16.68
Dermis225.38±29.72185.59±0.45121.85±0.56107.33±12.5680.21±12.0587.44±5.78
2h83.18±12.7971.33±12.385.08±1.226.72±1.848.43±0.8310.31±3.06
4h117.18±12.7988.13±20.048.87±2.358±1.3511.18±0.712±2.91
6h151.08±22.35108.77±14.3810.97±0.389.13±0.3812.51±3.9114.31±2.91
24h250.92±16.01132±14.9316.72±4.2213.23±4.2417.79±3.8422.41±1.56
S.c.stratumcorneum;eachaveragevaluewasobtainedfromthreeindependentrepetitions.

Figure1.Hesperidindistributionamongskinlayers:stratumcorneum,epidermis,anddermis(ng/cm2)afterapplication
and24hofincubationwithhesperidinsolutions:water(HeH2O,4μg/mL),ethanol(HeEtOH,4μg/mL,50%(v/v)),or
honeybushextracts:A,B,C,D.Eachaveragevaluewasobtainedfromthreeindependentrepetitions.Errorbarsrepresent
standarddeviations.Significantdifferencesamongsamplesaremarkedwithanasterisk(p<0.05).
Thequantitativeanalysisofhesperidindistributedamongskinlayersafteritsappli‐
cationfromsolutionsindicatedtheadvantageofhesperidinpermeationbothfromwater
andethanolsolutions.Thehighestamountofhesperidinwasdetectedinthedermisin
thecaseofbothsolutions,anditwasalmostfourtimeshigherthaninthestratum
corneum.Theresultswere225.38±29.72and185.59±0.45ng/cm2forthewaterandetha‐
nolsolutions,respectively.TheHPLCanalysisalsoshowedthatthehesperidinconcen‐
trationwasalmosttwotimeshigherintheepidermisthaninthestratumcorneumforboth
thesolutions(Table2).
Theadvantageofhesperidincumulationamongskinlayersaftertheapplicationof
solutionscomparedtohoneybushextracts(A,B,C,andD)wasevidenced.Theamount
ofhesperidininthestratumcorneum,epidermis,anddermiswashigherforwaterthan
forethanolsolution.
Intheexperimentswiththeextracts,thelowestamountofhesperidinwasforethanol
extractBinthestratumcorneum.Theconcentrationofhesperidinintheepidermisafter
honeybushextractswereappliedontheskinsurfacewascomparablewithaslightpre‐
dominanceofhesperidincumulationfromextractD.Amongthedermis,thedifferences
inhesperidincumulationweremorevisible:thelowesthesperidinconcentrationwas
Figure 1.
Hesperidin distribution among skin layers: stratum corneum, epidermis, and dermis (ng/cm
2
) after application
and 24 h of incubation with hesperidin solutions: water (HeH
2
O, 4
µ
g/mL), ethanol (HeEtOH, 4
µ
g/mL, 50% (v/v)), or
honeybush extracts: A, B, C, D. Each average value was obtained from three independent repetitions. Error bars represent
standard deviations. Significant differences among samples are marked with an asterisk (p< 0.05).
The quantitative analysis of hesperidin distributed among skin layers after its applica-
tion from solutions indicated the advantage of hesperidin permeation both from water and
ethanol solutions. The highest amount of hesperidin was detected in the dermis in the case
of both solutions, and it was almost four times higher than in the stratum corneum. The
results were 225.38
±
29.72 and 185.59
±
0.45 ng/cm
2
for the water and ethanol solutions,
respectively. The HPLC analysis also showed that the hesperidin concentration was almost
two times higher in the epidermis than in the stratum corneum for both the solutions
(Table 2).
The advantage of hesperidin cumulation among skin layers after the application of
solutions compared to honeybush extracts (A, B, C, and D) was evidenced. The amount of
hesperidin in the stratum corneum, epidermis, and dermis was higher for water than for
ethanol solution.
In the experiments with the extracts, the lowest amount of hesperidin was for ethanol
extract B in the stratum corneum. The concentration of hesperidin in the epidermis
after honeybush extracts were applied on the skin surface was comparable with a slight
predominance of hesperidin cumulation from extract D. Among the dermis, the differences
in hesperidin cumulation were more visible: the lowest hesperidin concentration was
detected for extracts C and D, whereas the hesperidin cumulation after the application of

Molecules 2021,26, 6547 5 of 16
extracts A and B exceeded 100 ng/cm
2
. Both for the solutions and extracts, the highest
concentration of hesperidin was detected in the dermis (Figure 1and Table 2).
2.2. Hesperidin Permeability through the Skin from Solutions and Honeybush Extracts
HPLC analysis revealed hesperidin’s ability to permeate through the skin to the
acceptor fluid (Figure 2).
Molecules2021,26,xFORPEERREVIEW5of16
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detectedforextractsCandD,whereasthehesperidincumulationaftertheapplicationof
extractsAandBexceeded100ng/cm2.Bothforthesolutionsandextracts,thehighest
concentrationofhesperidinwasdetectedinthedermis(Figure1andTable2).
2.2.HesperidinPermeabilitythroughtheSkinfromSolutionsandHoneybushExtracts
HPLCanalysisrevealedhesperidin’sabilitytopermeatethroughtheskintotheac‐
ceptorfluid(Figure2).

Figure2.Hesperidinpermeationthroughtheskinafter2,4,6,and24h(ng/cm2)fromtheapplication
ofhesperidinsolutions:water(HeH2O,4μg/mL),ethanol(HeEtOH,4μg/mL,50%(v/v)),orhoney‐
bushextracts:A,B,C,D.
Thepermeationofhesperidinafterapplicationontheskinsurfaceofhesperidinso‐
lutions—(HeH2O,HeEtOH)—wassimilarandwithinthelimitsofthestatisticalerrorin
thefirst2h.Thequantifiedanalysisshowedahighamountofhesperidinpresentedin
acceptorfluidafter2hfromapplication,whichindicatedtherelativelyquickpenetration
ofthecompoundthroughtheskin.Afurtherincreaseintheamountofhesperidinwas
observedafter4and6h.After24h,theHPLCanalysisshowedanamountofhesperidin
inthefluidatleasttwicehighercomparedtotheamountdeterminedafter2h(Table2).
Hesperidinpermeationthroughtheskin,aftertheapplicationofhoneybushextracts
ontheskinsurface,wasalsoobserved,anditincreasedduringthetimeofincubation.
After24hofhesperidin,theamountintheacceptorfluid(calculatedoncm2oftheskin)
washighestforextractD(22.41±1.56ng/cm2).Fortheremainingextracts,theobtained
amountsofthecompoundinthefluiddecreasedasfollows:C>A>B.
2.3.TransdermalDistributionofHesperidinwiththeUseofFluorescenceMicroscopy
Fluorescencemicroscopywasutilisedforconfirmationofthetransdermaldistribu‐
tionofhesperidinafter24hofincubationoftheethanolsolutiononthehumanskinsur‐
faceexvivo.Followedbycompletionofthepermeationexperiment,transverseskincuts
weremade.Formicroscopicanalysis,onlythepiecesofskinwithallvisiblelayersofthe
skinwereused.Freshlytakencross‐cuttingsofskinwereanalysedwithafluorescence
microscope(NikonEclipse50,filterUV2A,Ex330–380nm).Theresultsintheformof
photographsarepresentedinFigure3.Comparativeobservationoftheskinwithpure
Figure 2.
Hesperidin permeation through the skin after 2, 4, 6, and 24 h (ng/cm
2
) from the application
of hesperidin solutions: water (HeH
2
O, 4
µ
g/mL), ethanol (HeEtOH, 4
µ
g/mL, 50% (v/v)), or
honeybush extracts: A, B, C, D.
The permeation of hesperidin after application on the skin surface of hesperidin
solutions—(HeH
2
O, HeEtOH)—was similar and within the limits of the statistical error
in the first 2 h. The quantified analysis showed a high amount of hesperidin presented in
acceptor fluid after 2 h from application, which indicated the relatively quick penetration
of the compound through the skin. A further increase in the amount of hesperidin was
observed after 4 and 6 h. After 24 h, the HPLC analysis showed an amount of hesperidin
in the fluid at least twice higher compared to the amount determined after 2 h (Table 2).
Hesperidin permeation through the skin, after the application of honeybush extracts
on the skin surface, was also observed, and it increased during the time of incubation.
After 24 h of hesperidin, the amount in the acceptor fluid (calculated on cm
2
of the skin)
was highest for extract D (22.41
±
1.56 ng/cm
2
). For the remaining extracts, the obtained
amounts of the compound in the fluid decreased as follows: C > A > B.
2.3. Transdermal Distribution of Hesperidin with the Use of Fluorescence Microscopy
Fluorescence microscopy was utilised for confirmation of the transdermal distribution
of hesperidin after 24 h of incubation of the ethanol solution on the human skin surface
ex vivo. Followed by completion of the permeation experiment, transverse skin cuts
were made. For microscopic analysis, only the pieces of skin with all visible layers of the
skin were used. Freshly taken cross-cuttings of skin were analysed with a fluorescence
microscope (Nikon Eclipse 50, filter UV 2A, Ex 330–380 nm). The results in the form of
photographs are presented in Figure 3. Comparative observation of the skin with pure
ethanol (control, Figure 3A) and ethanolic hesperidin solution applied on the skin showed
a strong, bright yellow-green fluorescence in the analysed sample (Figure 3B). According

Molecules 2021,26, 6547 6 of 16
to the literature data, the observed fluorescence colour is characteristic for hesperidin [
66
].
The intensity of fluorescence was low in the area of the stratum corneum and epidermis,
while the dermis layer showed clearly visible yellow-green fluorescence. In this image
(Figure 3B), the strong fluorescence presented on the external surface of the skin indicated
that some part of hesperidin did not penetrate into the stratum corneum, and it was not
completely washed away after the end of the experiment. This hesperidin was probably
deposited on the surface of the skin.
Molecules2021,26,xFORPEERREVIEW6of16


ethanol(control,Figure3A)andethanolichesperidinsolutionappliedontheskinshowed
astrong,brightyellow‐greenfluorescenceintheanalysedsample(Figure3B).According
totheliteraturedata,theobservedfluorescencecolourischaracteristicforhesperidin[66].
Theintensityoffluorescencewaslowintheareaofthestratumcorneumandepidermis,
whilethedermislayershowedclearlyvisibleyellow‐greenfluorescence.Inthisimage
(Figure3B),thestrongfluorescencepresentedontheexternalsurfaceoftheskinindicated
thatsomepartofhesperidindidnotpenetrateintothestratumcorneum,anditwasnot
completelywashedawayaftertheendoftheexperiment.Thishesperidinwasprobably
depositedonthesurfaceoftheskin.

Figure3.Hesperidindistributioninthehumanskin(A—thecontrol,ethanol96%alone,B—ethanolhesperidinsolution
96%,0.01mg/mL).Ethanolandhesperidinsolutionswereappliedonthehumanskinexvivo.After24hofincubation,
skinwaswashedinwaterandanalysedunderafluorescencemicroscope,NikonEclipse50,filterUV2A,Ex330–380nm.
Thecomplexstructureoftheskinanditsmacromoleculesdetermineitsauto‐lumi‐
nescence,whichisvisibleintheleftimage(Figure3A,skinsampleonwhichonlyethanol
wasapplied,controlsample)[67].Theobtainedpicturesindicatethathesperidinenhances
skinfluorescence.
Thefluorescencemicroscopyresultsconfirmthequantitativeanalysisofthetrans‐
dermaldistributionofhesperidinafteritsapplicationandincubationontheskinsurface
(Table2).Thefluorescencewasnoticeableespeciallyintheareaofthedermiswhere,ac‐
cordingtotheresultspresentedinFigure1,hesperidinunderwentthehighestabsorption.
However,accordingtoXieetal.,hesperetin,theaglyconofhesperidin,interactswithpro‐
teins,affectingwithslightfluorescencethequenchingofmacromolecules[68].Aninter‐
actioncouldbecausedbybothhydrophobicandelectrostaticbondsbetweenproteinsand
hesperetin.Itwasindicatedthathydroxylgroupsarethemostimportantintheproton
transversion[69].Hesperetin,whichinteractswithproteins,canbestoredandfloated
withthem[68].Ourcomparisondataofthecontrolandintenseyellow‐greenfluorescence
presentedintherightpicture(Figure3B)suggestthatnonbondedhesperidinwasrespon‐
sibleforthebrightfluorescence.Aprobablyunknownamountofhesperidininteracted
withmacromoleculesoftheepidermisordermis,resultinginquenching.However,the
amountofnoninteractedhesperidinwasstillenoughtogivevisiblefluorescenceinthe
dermis.ThefreehesperidinpresentedinthedermislayerisneededtoprotectECMmac‐
romoleculesfrominternalandexternaloxidativestress[14].

Figure 3.
Hesperidin distribution in the human skin (
A
—the control, ethanol 96% alone,
B
—ethanol hesperidin solution
96%, 0.01 mg/mL). Ethanol and hesperidin solutions were applied on the human skin ex vivo. After 24 h of incubation, skin
was washed in water and analysed under a fluorescence microscope, Nikon Eclipse 50, filter UV 2A, Ex 330–380 nm.
The complex structure of the skin and its macromolecules determine its auto-luminescence,
which is visible in the left image (Figure 3A, skin sample on which only ethanol was applied,
control sample) [67]. The obtained pictures indicate that hesperidin enhances skin fluorescence.
The fluorescence microscopy results confirm the quantitative analysis of the trans-
dermal distribution of hesperidin after its application and incubation on the skin surface
(Table 2)
. The fluorescence was noticeable especially in the area of the dermis where, ac-
cording to the results presented in Figure 1, hesperidin underwent the highest absorption.
However, according to Xie et al., hesperetin, the aglycon of hesperidin, interacts with
proteins, affecting with slight fluorescence the quenching of macromolecules [
68
]. An
interaction could be caused by both hydrophobic and electrostatic bonds between proteins
and hesperetin. It was indicated that hydroxyl groups are the most important in the proton
transversion [
69
]. Hesperetin, which interacts with proteins, can be stored and floated with
them [
68
]. Our comparison data of the control and intense yellow-green fluorescence pre-
sented in the right picture (Figure 3B) suggest that nonbonded hesperidin was responsible
for the bright fluorescence. A probably unknown amount of hesperidin interacted with
macromolecules of the epidermis or dermis, resulting in quenching. However, the amount
of noninteracted hesperidin was still enough to give visible fluorescence in the dermis. The
free hesperidin presented in the dermis layer is needed to protect ECM macromolecules
from internal and external oxidative stress [14].
2.4. Comparison of Mangiferin Permeation into the Skin and Permeability through the Skin from
Solutions and Honeybush Extracts
The ability of mangiferin to cross the skin stratum corneum barrier was estimated
and demonstrated in our previous paper [
40
] where we analysed both the mangiferin
distribution among skin layers and the permeation through the skin to the acceptor fluid,
after application of mangiferin solutions (MH
2
OH, MEtOH) on the skin surface. In this

Molecules 2021,26, 6547 7 of 16
study, we additionally estimated the distribution and permeation of the honeybush extracts
(A, B, C, D) through the skin.
HPLC analysis with electrochemical detection indicated that mangiferin is capable
of entering into and crossing through the stratum corneum, after the application of both
mangiferin solutions and honeybush extracts (Table 3and Figure 4).
Table 3.
Accumulation of mangiferin analysed by HPLC-UV among skin layers: stratum corneum, epidermis, and dermis,
and mangiferin permeation through the skin after 2, 4, 6, and 24 h (ng/cm
2
) from the application of mangiferin solutions
(MH2O, MEtOH) or honeybush extracts (A, B, C, D).
MH2O MEtOH Extract A Extract B Extract C Extract D
S.c. 32.62 ±4.61 29.92 ±2.07 24.46 ±2.36 29.44 ±7.51 56.92 ±3.72 26.92 ±1.53
Epidermis 41.62 ±7.45 32.08 ±1.97 32.74 ±2.45 48.51 ±7.08 74.26 ±5.95 32.72 ±2.74
Dermis 53.32 ±16.41 35.23 ±4.73 23.95 ±2.15 29.13 ±1,84 58.67 ±2.67 24.1 ±1.97
2 h 30.23 ±5.22 48.85 ±7.37 30.97 ±1.54 33.85 ±5.31 61.03 ±5.05 34.1 ±7.44
4 h 35.38 ±5.9 59.08 ±6.76 39.95 ±1.72 42.31 ±8.18 82.36 ±7.27 43.28 ±6.74
6 h 40.54 ±7.77 69.15 ±7 49.23 ±2.66 50.92 ±7.52 111.54 ±9.39 56.56 ±4.66
24 h 66.15 ±11.92 77.78 ±8.38 59.33 ±3.76 62.82 ±9.46 152.36 ±8.57 75.69 ±2.14
S.c. stratum corneum; each average value was obtained from three independent repetitions.
Molecules2021,26,xFORPEERREVIEW7of16
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
2.4.ComparisonofMangiferinPermeationintotheSkinandPermeabilitythroughtheSkinfrom
SolutionsandHoneybushExtracts
Theabilityofmangiferintocrosstheskinstratumcorneumbarrierwasestimated
anddemonstratedinourpreviouspaper[40]whereweanalysedboththemangiferindis‐
tributionamongskinlayersandthepermeationthroughtheskintotheacceptorfluid,
afterapplicationofmangiferinsolutions(MH2OH,MEtOH)ontheskinsurface.Inthis
study,weadditionallyestimatedthedistributionandpermeationofthehoneybushex‐
tracts(A,B,C,D)throughtheskin.
HPLCanalysiswithelectrochemicaldetectionindicatedthatmangiferiniscapableof
enteringintoandcrossingthroughthestratumcorneum,aftertheapplicationofboth
mangiferinsolutionsandhoneybushextracts(Table3andFigure4).
Table3.AccumulationofmangiferinanalysedbyHPLC‐UVamongskinlayers:stratumcorneum,epidermis,anddermis,
andmangiferinpermeationthroughtheskinafter2,4,6,and24h(ng/cm2)fromtheapplicationofmangiferinsolutions
(MH2O,MEtOH)orhoneybushextracts(A,B,C,D).
MH2OMEtOHExtractAExtractBExtractCExtractD
S.c.32.62±4.6129.92±2.0724.46±2.3629.44±7.5156.92±3.7226.92±1.53
Epidermis41.62±7.4532.08±1.9732.74±2.4548.51±7.0874.26±5.9532.72±2.74
Dermis53.32±16.4135.23±4.7323.95±2.1529.13±1,8458.67±2.6724.1±1.97
2h30.23±5.2248.85±7.3730.97±1.5433.85±5.3161.03±5.0534.1±7.44
4h35.38±5.959.08±6.7639.95±1.7242.31±8.1882.36±7.2743.28±6.74
6h40.54±7.7769.15±749.23±2.6650.92±7.52111.54±9.3956.56±4.66
24h66.15±11.9277.78±8.3859.33±3.7662.82±9.46152.36±8.5775.69±2.14
S.c.stratumcorneum;eachaveragevaluewasobtainedfromthreeindependentrepetitions.

Figure4.Mangiferindistributionamongskinlayers:stratumcorneum,epidermis,anddermis(ng/cm2)afterapplication
and24hofincubationofmangiferinsolutions:water(MH2O,25μg/mL),ethanol(25μg/mL,MEtOH,50%(v/v)),orhon‐
eybushextracts:A,B,C,D.Eachaveragevaluewasobtainedfromthreeindependentrepetitions.Errorbarsrepresent
standarddeviations.Significantdifferencesamongsamplesaremarkedwithanasterisk(p<0.05).
Thehighestamountofmangiferinthatenteredintoandpermeatedthroughthestra‐
tumcorneumispresentedforextractC,whichisthemostpromisingsourceofmangiferin
forskinpermeation.Thecompoundaccumulationwashighestintheepidermis,while,in
thestratumcorneumanddermis,theamountofdeterminedmangiferinwascomparable
(Table3).Thesecondsignificantamountofmangiferinintransdermaldistributionwas
Figure 4. Mangiferin distribution among skin layers: stratum corneum, epidermis, and dermis (ng/cm2) after application
and 24 h of incubation of mangiferin solutions: water (MH
2
O, 25
µ
g/mL), ethanol (25
µ
g/mL, MEtOH, 50% (v/v)), or
honeybush extracts: A, B, C, D. Each average value was obtained from three independent repetitions. Error bars represent
standard deviations. Significant differences among samples are marked with an asterisk (p< 0.05).
The highest amount of mangiferin that entered into and permeated through the stra-
tum corneum is presented for extract C, which is the most promising source of mangiferin
for skin permeation. The compound accumulation was highest in the epidermis, while, in
the stratum corneum and dermis, the amount of determined mangiferin was comparable
(Table 3). The second significant amount of mangiferin in transdermal distribution was
observed after the application of MH
2
O (Table 3and Figure 4). In this case, the mangiferin
cumulation was highest in the dermis.

Molecules 2021,26, 6547 8 of 16
The concentration of mangiferin detected in the stratum corneum was similar to each
other with only slight differences: MH
2
O > MEtOH > B > D > A (Table 3). However,
the highest amount was detected for extract C. In the epidermis, the lowest concentra-
tion of mangiferin was estimated for extracts A and D and ethanol solution MEtOH,
while mangiferin from extract B and MH
2
O indicated a more significant accumulation of
the compound. In the dermis, differences in mangiferin concentration were as follows:
C > MH
2
O > MEtOH > B > D = A. The lowest accumulation of mangiferin was detected
among skin layers after the application of extracts A and D.
The mangiferin permeation through the skin from all analysed solutions and honey-
bush extracts was constant and occurred relatively quickly (Figure 5). The most effective
permeation of mangiferin into the acceptor fluid, during the 24 h of analysis, was observed
for extract C, while the least was for MH
2
O, extracts A and B. The mangiferin permeation
process for extracts A and B was similar, and a slight increase was observed for the MEtOH
solution and extract D.
Molecules2021,26,xFORPEERREVIEW8of16


observedaftertheapplicationofMH2O(Table3andFigure4).Inthiscase,themangiferin
cumulationwashighestinthedermis.
Theconcentrationofmangiferindetectedinthestratumcorneumwassimilartoeach
otherwithonlyslightdifferences:MH2O>MEtOH>B>D>A(Table3).However,the
highestamountwasdetectedforextractC.Intheepidermis,thelowestconcentrationof
mangiferinwasestimatedforextractsAandDandethanolsolutionMEtOH,whileman‐
giferinfromextractBandMH2Oindicatedamoresignificantaccumulationofthecom‐
pound.Inthedermis,differencesinmangiferinconcentrationwereasfollows:C>MH2O
>MEtOH>B>D=A.Thelowestaccumulationofmangiferinwasdetectedamongskin
layersaftertheapplicationofextractsAandD.
Themangiferinpermeationthroughtheskinfromallanalysedsolutionsandhoney‐
bushextractswasconstantandoccurredrelativelyquickly(Figure5).Themosteffective
permeationofmangiferinintotheacceptorfluid,duringthe24hofanalysis,wasobserved
forextractC,whiletheleastwasforMH2O,extractsAandB.Themangiferinpermeation
processforextractsAandBwassimilar,andaslightincreasewasobservedfortheME‐
tOHsolutionandextractD.

Figure5.Mangiferinpermeationthroughtheskinafter2,4,6,and24h(ng/cm2)fromtheapplicationofmangiferinsolu‐
tions:water(MH2O,25μg/mL),ethanol(MEtOH,25μg/mL,50%(v/v)),orhoneybushextracts:A,B,C,D.
3.Discussion
Thefirststageintheprocessofxenobioticpenetrationintotheskinandpermeation
throughtheskinisovercomingthestratumcorneumbarrier,whichistreatedasasemi‐
permeablemembrane.Bothanalysedcompoundsinourstudy—mangiferinandhesperi‐
din—havelogPbetween1and3(hesperidin:1.78,mangiferin:2.73);themangiferinmolar
massdoesnotexceed500Da(422.34g/mol),thoughthehesperidinmolarmasishigher
withthevalueof610.19g/mol.Thissuggeststhatmangiferinandhesperidinshouldbe
abletopassthestratumcorneum[12,70,71].Thelimitingfactorforbothpolyphenolscould
betheirlowwatersolubilityandabranchedstructure[72,73].Ouranalysisrevealedthe
abilityofmangiferinandhesperidintopermeatethestratumcorneumlayerfromboth
honeybushextractsandsolutions,withapredominanceofhesperidinfromthewaterso‐
lutionandmangiferinfromtheethanolextractofgreenhoneybush(extractC).Bothcom‐
poundspresentedarelativelyfastabilitytopermeatethroughtheskin,especiallyfrom
appliedsolutionsforhesperidinandextractCformangiferin.Thesesolutionsandthe
extractappliedontheskinexhibitedthehighestcumulationofanalysedcompounds
Figure 5.
Mangiferin permeation through the skin after 2, 4, 6, and 24 h (ng/cm
2
) from the application of mangiferin
solutions: water (MH2O, 25 µg/mL), ethanol (MEtOH, 25 µg/mL, 50% (v/v)), or honeybush extracts: A, B, C, D.
3. Discussion
The first stage in the process of xenobiotic penetration into the skin and perme-
ation through the skin is overcoming the stratum corneum barrier, which is treated as
a semi-permeable membrane. Both analysed compounds in our study—mangiferin and
hesperidin—have logP between 1 and 3 (hesperidin: 1.78, mangiferin: 2.73); the mangiferin
molar mass does not exceed 500 Da (422.34 g/mol), though the hesperidin molar mas is
higher with the value of 610.19 g/mol. This suggests that mangiferin and hesperidin should
be able to pass the stratum corneum [
12
,
70
,
71
]. The limiting factor for both polyphenols
could be their low water solubility and a branched structure [
72
,
73
]. Our analysis revealed
the ability of mangiferin and hesperidin to permeate the stratum corneum layer from
both honeybush extracts and solutions, with a predominance of hesperidin from the water
solution and mangiferin from the ethanol extract of green honeybush (extract C). Both
compounds presented a relatively fast ability to permeate through the skin, especially from
applied solutions for hesperidin and extract C for mangiferin. These solutions and the ex-
tract applied on the skin exhibited the highest cumulation of analysed compounds among
skin layers, which make them the most promising sources of mangiferin and hesperidin
release into the skin and through the skin.

Molecules 2021,26, 6547 9 of 16
Surprisingly, the lowest hesperidin amount from both solutions was accumulated in
the stratum corneum, and the highest amount was detected in the dermis. Our analysis
showed that hesperidin sourced from the water solution penetrated into the skin and
permeated through the skin with an advantage over hesperidin from the ethanol solution.
These data could indicate that for hesperidin, ethanol is not a sorption promoter [
74
].
Despite the lower hesperidin solubility in water [
72
], a higher amount of this polyphenol
can permeate to the dermis after the application of a water solution on the skin. The dermis,
as the largest layer of the skin, is composed of sensitive to oxidative stress proteins, and
the presence of hesperidin, a compound with strong antioxidant properties, can work as
effective protection against macromolecules [52].
The confirmation of hesperidin cumulation among skin layers and the interaction
of hesperidin molecules was observed with fluorescence microscopy. The experiment
confirmed that a lower yellow-green fluorescence assigned to hesperidin could be seen
in the stratum corneum and epidermis, while the distribution of the xenobiotic in the
dermis was brighter. The intensity of fluorescence in the epidermis and dermis after
hesperidin application proved the presence of free hesperidin, which did not interact with
skin macromolecules [
68
]. The permanent hesperidin interaction with molecules of the
dermis extracellular matrix (ECM) was generally slight, though the mechanism of the
protection can differ from the conventional antioxidant trail [
75
–
77
]. Nevertheless, the
experiment was carried out ex vivo, and it should be emphasised that in a living organism,
the partition of free hesperidin and that associated with macromolecules could be different.
The ECM is the largest element of healthy skin and is responsible for homeostasis and the
preservation of the mechanical and biological properties of the skin. It is highly complex
system, the protection of which by antioxidants is thus essential [21,68].
It should be emphasised that fluorescence techniques are used to determine transder-
mal drug uptake, though they could be utilised only in the case of molecules with fluores-
cence properties that have different wavelengths than skin macromolecules [
40
,
67
,
78
,
79
].
As it was indicated in our research, hesperidin with its fluorescence properties can be
successfully analysed with these techniques [
66
]. However, the visualisation of the trans-
dermal distribution of hesperidin from honeybush extracts was impossible due to a large
number of different polyphenols with fluorescence abilities presented in the extracts [17].
The present study also included the analysis of hesperidin permeation from the skin
after the application of hesperidin solutions during the 24 h. The research indicated
that hesperidin occurred in the acceptor fluid relatively quickly, especially after the first
two hours. During the following hours, hesperidin permeation was constantly occurring
despite the solution used, with the advantage of water solution. The differences between the
permeability of hesperidin derived from water and ethanol solution were seen especially
after 24 h of the analysis. The experimental data obtained in this part confirmed that
hesperidin from water solution has a better ability to penetrate into the skin and permeate
through the skin than hesperidin can from ethanol solution.
We also analysed hesperidin permeation to the skin and through the skin after appli-
cation of the honeybush extracts on the skin surface. The experiments showed the best
accumulation of hesperidin in the dermis, also from the extracts obtained with water. These
results indicate that, regardless of the solution or extracts applied on the skin, hesperidin
permeates in a higher amount to the dermis from the water carrier. There is also a slight
advantage in the penetration into the skin of hesperidin from water-unfermented extracts
compared to fermented honeybush (A > B).
A major disadvantage of using plant material is the high variability in chemical
composition and antioxidant properties, which depends not only on the changing envi-
ronmental conditions, but also on the time of harvest, as well as storage and transport
conditions [
20
,
80
–
82
]. If we consider polyphenolic fractions as a source of antioxidants
in the skin layers, it is important to estimate “competition” and “enhancer” properties of
chemicals [15,16]. Honeybush extracts contain many biologically active molecules, which
could potentially improve or inhibit the penetration of the most quantitative compounds

Molecules 2021,26, 6547 10 of 16
into the skin: mangiferin and hesperidin [
83
]. In the case of both compounds, no compari-
son analysis of the cumulation into the skin or permeation through the skin from solutions
and plant extracts has been performed so far.
Despite the applied solutions or honeybush extracts on the skin surface, mangiferin
presented a lower ability than hesperidin to penetrate into the skin. Ethanol extract from
the green honeybush (extract C) applied on the human skin surface showed the highest
mangiferin penetration into the skin and the fastest ability to permeate to the acceptor
fluid during the 24 h of conducted analysis. There was also no evidence of an advantage
in accumulation and permeation studies of mangiferin from solutions than mangiferin
from honeybush extracts. The water solution of mangiferin, similar to hesperidin solution,
indicated a higher ability for transdermal distribution than ethanol solution. Our study
showed that mangiferin has a limited ability to pass the stratum corneum barrier and to
obtain higher concentrations among skin layers.
However, the above results of mangiferin permeation from honeybush extracts (espe-
cially from nonfermented ethanol—C) are generally promising, especially considering the
fact that more mangiferin was absorbed from the ethanol extract than from solutions with
higher concentrations of the compound. The obtained data of mangiferin permeation do
not give an unambiguous answer that ethanol could be a sorption promoter for mangiferin
delivered into the skin from honeybush extracts. The synergism of biological activity of
polyphenolic compounds in this plant species may be responsible for this effect [
84
,
85
].
In addition, the strengthening action of hesperidin on mangiferin has been previously
observed [29,83].
In conclusion, mangiferin and hesperidin were detected in all skin layers and in the
acceptor fluid in the form of both solutions and extracts applied on the human skin surface.
The analysis revealed the predominance of the transdermal cumulation of hesperidin over
mangiferin. On the other hand, permeation through the skin was better for mangiferin
than for hesperidin. Our ex vivo study on mangiferin showed that its permeation into
and through the skin is significantly higher for the ethanol-nonfermented extract than
for solutions. The hesperidin transdermal distribution and permeation study on Cyclopia
sp. extracts are described in this work for the first time. The results clearly indicated
that hesperidin solutions and mangiferin, originating from the ethanol extract of “green”
honeybush, can be a source of transdermal release of the analysed molecules, which have
strong protective and regenerative properties in the skin. The compounds may be used as
antioxidants in dermatology and in topical ways of drug administration, especially due to
the limited oral absorption of both polyphenols.
4. Materials and Methods
4.1. Materials
Mangiferin, hesperidin, and sodium azide were purchased from Sigma Chemical Co.
(St. Louis, MO, USA). HPLC-grade methanol and ethanol were purchased from P.O.Ch.
(Gliwice, Poland).
4.2. Plant Material and Extract Preparation
Plant material was composed of dried leaves and branches declared as the mixtures
of different honeybush species (Cyclopia sp.). Nonfermented (“green”) Cyclopia sp. material
was purchased from Ukajali Marcin Majka Co., (Krakow, Poland) for preparation of extracts
A and C. Fermented Cyclopia sp. plant material was purchased from KAWON-HURT Co.,
(Gostyn, Poland) for preparation of extracts B and D. Plant material and used solvents
are summarised in Table 1. Representative samples of each plant material were deposited
at the Department of Biology and Pharmaceutical Botany, Medical University of Gdansk,
Poland. Extract preparation: 15 g of each plant material was mechanically homogenised
to obtain a particle size < 2.0 mm. Extraction with 100 mL of water or ethanol (50%, v/v)
was performed three times with the use of an ultrasonic water bath (50 Hz) for 30 min at
90
◦
C. After filtration through Whattman filter paper (390
µ
m pore size), obtained extracts

Molecules 2021,26, 6547 11 of 16
were evaporated under vacuum at 60
◦
C. The dry residue was redissolved in either water
or 50% (v/v) ethanol to the final concentration of 30 mg/mL. For the quantitative analysis
of mangiferin and hesperidin in the resulting extracts, HPLC with Photodiode Array
Coulometric and Coulometric Electrochemical Array Detection was used.
4.3. Penetration and Permeation Studies
The studies were performed with the use of human cadaver full skin. Before the
experiment, the skin was collected from the region of the thorax of six 40–60-years-old
donors, and stored frozen at
−
20
◦
C. The solutions and extracts were applied as an
infinite dose: mangiferin and hesperidin, and ethanol (50%, v/v) and water solutions at
concentrations of 25
µ
g/mL and 4
µ
g/mL, respectively; and extracts A–D at a concentration
of 30 mg/mL. The ethanol at a concentration of 50% (v/v) was applied on the skin as a
blank control. The skin (thickness—0.75 mm; diffusion area—0.65 cm
2
) was placed in
the flow-through Bronaugh diffusion cell apparatus (Figure 6). The static diffusion cell is
composed of two chambers: donor and acceptor. Between these chambers, a part of skin
is placed. The analysed sample is applied on the upper skin surface and the dermis is in
constant contact with homogenised and circulated acceptor fluid. The permeant from the
skin can be taken to the analysis at a specific time period.
Molecules2021,26,xFORPEERREVIEW11of16


ofGdansk,Poland.Extractpreparation:15gofeachplantmaterialwasmechanicallyho‐
mogenisedtoobtainaparticlesize<2.0mm.Extractionwith100mLofwaterorethanol
(50%,v/v)wasperformedthreetimeswiththeuseofanultrasonicwaterbath(50Hz)for
30minat90°C.AfterfiltrationthroughWhattmanfilterpaper(390μmporesize),ob‐
tainedextractswereevaporatedundervacuumat60°C.Thedryresiduewasredissolved
ineitherwateror50%(v/v)ethanoltothefinalconcentrationof30mg/mL.Forthequan‐
titativeanalysisofmangiferinandhesperidinintheresultingextracts,HPLCwithPhoto‐
diodeArrayCoulometricandCoulometricElectrochemicalArrayDetectionwasused.
4.3.PenetrationandPermeationStudies
Thestudieswereperformedwiththeuseofhumancadaverfullskin.Beforetheex‐
periment,theskinwascollectedfromtheregionofthethoraxofsix40–60‐years‐olddo‐
nors,andstoredfrozenat−20°C.Thesolutionsandextractswereappliedasaninfinite
dose:mangiferinandhesperidin,andethanol(50%,v/v)andwatersolutionsatconcentra‐
tionsof25μg/mLand4μg/mL,respectively;andextractsA–Dataconcentrationof30
mg/mL.Theethanolataconcentrationof50%(v/v)wasappliedontheskinasablank
control.Theskin(thickness—0.75mm;diffusionarea—0.65cm
2
)wasplacedintheflow‐
throughBronaughdiffusioncellapparatus(Figure6).Thestaticdiffusioncelliscomposed
oftwochambers:donorandacceptor.Betweenthesechambers,apartofskinisplaced.
Theanalysedsampleisappliedontheupperskinsurfaceandthedermisisinconstant
contactwithhomogenisedandcirculatedacceptorfluid.Thepermeantfromtheskincan
betakentotheanalysisataspecifictimeperiod.

Figure6.Diffusioncellapparatus.
Salinesolution,20mL(with0.005%sodiumazide),wasrecirculatedbeneaththeskin
withaconstantrateof10mL/h.Acceptorfluidensuredthesinkcondition.Solutionsor
extractswereappliedonthesurfaceoftheskinandleftfor24h.Thechambersystemwas
incubatedatthetemperatureof37°C±0.5°C.After2,4,6,and24hfromtheapplication
ofpenetrants,samplesofacceptorfluidwerecollected.Beforeskinlayers’disconnection,
thepenetrantswereremovedfromtheskin.Thestratumcorneumwasseparatedbythe
tape‐strippingmethod,using30fragmentsofa3madhesivetapewiththefollowingpa‐
rameters:pressure~1kg/cm
2
(appliedbystamp),2sdurationofpressure,andarapid
removalrateatanangleof45°.Theepidermisanddermiswereisolatedbytheheatsep‐
arationtechnique[86].Thewholeskinwasimmersedinwaterat60°Cfor45s;afterward,
theskinlayersweredividedbytweezers.Allskinlayerswereextractedwithmethanol
andtheobtainedsolutionswereanalysedbyHPLC.
TheanalysiswasundertherevisionandapprovaloftheIndependentBioethicsCom‐
missionforResearchoftheMedicalUniversityofGdansk(numberNKBBN/120‐41/2014).
Thepermeabilityandpenetrationdatawereexpressedasmeanvalues±standard
deviation(SD).Statisticalcomparisonsoftheresultswereanalysedwithatwo‐way
ANOVAwithposthocTukeytest(p<0.05).
Figure 6. Diffusion cell apparatus.
Saline solution, 20 mL (with 0.005% sodium azide), was recirculated beneath the skin
with a constant rate of 10 mL/h. Acceptor fluid ensured the sink condition. Solutions or
extracts were applied on the surface of the skin and left for 24 h. The chamber system
was incubated at the temperature of 37
◦
C
±
0.5
◦
C. After 2, 4, 6, and 24 h from the
application of penetrants, samples of acceptor fluid were collected. Before skin layers’
disconnection, the penetrants were removed from the skin. The stratum corneum was
separated by the tape-stripping method, using 30 fragments of a 3 m adhesive tape with
the following parameters: pressure ~1 kg/cm
2
(applied by stamp), 2 s duration of pressure,
and a rapid removal rate at an angle of 45
◦
. The epidermis and dermis were isolated by the
heat separation technique [
86
]. The whole skin was immersed in water at 60
◦
C for 45 s;
afterward, the skin layers were divided by tweezers. All skin layers were extracted with
methanol and the obtained solutions were analysed by HPLC.
The analysis was under the revision and approval of the Independent Bioethics Commis-
sion for Research of the Medical University of Gdansk (number NKBBN/120-41/2014).
The permeability and penetration data were expressed as mean values
±
standard
deviation (SD). Statistical comparisons of the results were analysed with a two-way ANOVA
with post hoc Tukey test (p< 0.05).
4.4. Chromatography
The HPLC system and analysis condition were described previously [
29
]. Briefly,
the HPLC system was equipped with a spectrophotometric diode array 340S detector
pump P 580, column thermostat STH 585, and automated sample injector ASI-100 (Dionex
Corporation, Sunnyvale, CA, USA). In addition, for the results confirmation, an additional

Molecules 2021,26, 6547 12 of 16
detector was utilised: Coulochem II electrochemical detector with a 5020 model guard and
a 5010 model analytical cell (ESA, Chelmsford, MA, USA) operated by the Chromeleon
chromatography-management system v. 6.8 (Dionex). The separation of mangiferin and
hesperidin was performed on a Hypersil Gold C
18
column (150 mm
×
4.6 mm I.D., 5
µ
m
particle) equipped with a Hypersil Gold guard column (10 mm
×
4.6 mm I.D., 5
µ
m
particle) (Thermo Electron Corporation, Dreieich, Germany).
Before use, the isocratic mobile phase (15 mM of sodium phosphate, pH 4.0 with
85% orthophosphoric acid and acetonitrile (65/35 (v/v)) was filtered (0.22
µ
m membrane
filter) and degassed by vacuum. The flow rate was maintained at 1.0 mL/min and the
injection volume was 20
µ
L. The temperature in the column was 20
◦
C and that in the
automated sample injector was set at 8
◦
C. To establish the electrochemical behaviour of
mangiferin and hesperidin, repeated injections of working standard solutions (0.1 mg/mL)
and detection at potentials from
−
0.5 to +1.2 V were studied [
87
]. Both mangiferin and
hesperidin exhibited good responses of hydrodynamic voltammograms in the ranges from
+0.35 to +0.95 V. The applied potentials on the guard cell, electrode E1, and electrode E2
were +1.1 V, +0.35 V, and +0.95 V, respectively. The photodiode array detector was set at
225, 254, 280, and 360 nm wavelengths, respectively [83].
Mangiferin and hesperidin exhibited a good linearity of calibration curves in the in-
vestigated ranges (10.0–100.0 ng, R
2
> 0.9995). Limits of detection (LOD) and quantification
(LOQ) for mangiferin and hesperidin were 0.35 and 9.7, and 0.24 and 8.6 ng/mL, respec-
tively. Precision and reproducibility were estimated from six consecutive replicates, and the
R.S.D. values for mangiferin and hesperidin were lower than 0.9% and 3.7%, respectively.
4.5. Fluorescence Microscopy
The utilisation of fluorescence microscopy to confirm the distribution of mangiferin
in the different skin layers was described by Ochocka et al., 2017 [
40
]. Both mangiferin
and hesperidin are substances having fused heterocyclic rings in their structure, which
determine their ability to fluoresce [
88
,
89
]. The excitation and emission maxima of hes-
peridin are 350 nm and 420 nm, respectively [
66
]. Biomolecules such as NADPH, elastin,
and collagen also exhibit fluorescence [
67
], which makes observation much more difficult
because interactions between chemical compounds having chromophores can enhance or
quench fluorescence [90].
To confirm the transdermal distribution of hesperidin with the use of fluorescence
microscopy, an additional experiment was performed. The hesperidin solution applied on
the skin was 10
µ
g/mL (96% EtOH (v/v)); in the control study, only ethanol was used (96%
EtOH (v/v)). The course of the research was analogous to that in the permeation study, but
only without collecting samples from the outlet and separation of the skin layers after the
experiment. Skin removed from the Bronaught cells was frozen at
−
60
◦
C in a Cryotome
E (Thermo Electron Corporation, Rugby, UK). Just before fluorescence microscopy, several
cross-cuttings of the frozen skin were performed in order to obtain the transverse profile of the
skin. Sections with all visible layers of the skin profile were placed on a laboratory glass slide.
Freshly thawed skin cuttings were analysed with a fluorescence microscope (Nikon Eclipse
50 with filter UV 2A: 330 nm excitation, 380 nm emission, equipped with the lamp, Nikon
super-high-pressure mercury, and the camera, Nikon 05–5MC). The analysis of the obtained
images (magnification 100 times) was performed in the program NIS Elements AR3.2.
Author Contributions:
Conceptualization, A.H.; Data curation, J.R.O., K.C. and J.S.-H.; Formal anal-
ysis, H.B. and K.C.; Funding acquisition, J.R.O.; Investigation, A.H.; Methodology, A.H., J.R.O. and
H.B.; Project administration, K.C. and J.S.-H.; Supervision, J.R.O.; Validation, J.S.-H.; Visualization,
K.C.; Writing—original draft, A.H. and J.S.-H. All authors have read and agreed to the published
version of the manuscript.
Funding: This research recived no external funding.

Molecules 2021,26, 6547 13 of 16
Institutional Review Board Statement:
The analysis was under the revision and approval of the
Independent Bioethics Commission for Research of the Medical University of Gdansk (number
NKBBN/120-41/2014).
Informed Consent Statement:
The studies were conducted post mortem (ex vivo). For this type of
research in Poland, the only document required is the consent of the Bioethics Commission. Researchers
responsible for this stage of the work have an indefinite consent (number NKBBN/120-41/2014).
Data Availability Statement: All data are included in the manuscript.
Acknowledgments:
The authors would like to thank the Medical University of Gda´nsk for the
financial support of the study (statutory funds).
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.
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