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ORIGINAL ARTICLES
Departamento de Farmácia1, Universidade Federal dos Vales do Jequitinhonha e Mucuri, Brasil;
Curso de Pós-graduac¸ão em Ciências Farmacêuticas (CiPharma)2, Escola de Farmácia, Universidade Federal de
Ouro Preto, Brasil
In vivo wound healing effects of Symphytum officinale L. leaves extract in
different topical formulations
L. U. Araújo1, P. G. Reis2, L. C. O. Barbosa2, D. A. Saúde-Guimarães2, A. Grabe-Guimarães 2, V. C. F. Mosqueira2,
C. M. Carneiro 2, N. M. Silva-Barcellos2
Received April 15, 2011, accepted June 30, 2011
Prof. Dr. Neila Márcia Silva Barcellos, Rua Costa Sena n◦171, Centro, CEP: 35 400-000, Ouro Preto, Minas Gerais,
Brasil
neila@ef.ufop.br
Pharmazie 67: 355–360 (2012) doi: 10.1691/ph.2012.1563
The present work evaluates wound healing activity of leaves extracts of Symphytum officinale L. (comfrey)
incorporated in three pharmaceutical formulations. Wound healing activity of comfrey was determined by
qualitative and quantitative histological analysis of open wound in rat model, using allantoin as positive
control. Three topical formulations, carbomer gel, glycero-alcoholic solution and O/W emulsion (soft lotion)
were compared. The histological analysis of the healing process shows significant differences in treatment,
particularly on its intensity and rate. The results indicate that emulsion containing both extracts, commercial
and prepared, induced the largest and furthest repair of damaged tissue. This could be evidenced from
day 3 to 28 by increase in collagen deposition from 40% to 240% and reduction on cellular inflammatory
infiltrate from 3% to 46%. However, 8% prepared extract in emulsion presented the best efficacy. This work
clearly demonstrates that comfrey leaves have a wound healing activity. The O/W emulsion showed to be
the vehicle most effective to induce healing activity, particularly with extracts obtained from comfrey leaves
collected in Minas Gerais state in Brazil. It shows the best efficacy to control the inflammatory process and
to induce collagen deposition at 8% concentration.
1. Introduction
Symphytum officinale L., comfrey, Boraginaceae, has been con-
sidered in traditional medicine for its anti-inflammatory (Predel
et al. 2005), analgesic (Goldman et al. 1985; Kucera et al. 2004;
Grube et al. 2007), anti-edematous (Kucera et al. 2004) and
adstringent properties (Staiger 2007). For over 2000 years, it
has been widely used by the population to treat a variety of ail-
ments and to favor the growth of new tissues in wounds and bone
fractures (Staiger 2007). Additionally, numerous compounds
obtained from comfrey like mucilage, allantoin, alkaloids, tan-
nins and sugars, have had their biological activities documented
(Youngken 1950). The wound healing action of comfrey has
been attributed to the presence of allantoin (Saito and Oliveira
1986; Martindale 2002; Cunha et al. 2003; Carvalho 2004),
Anti-irritating, hydratating and anti-inflammatory properties are
atributed to mucilage (Saito and Oliveira 1986; Cunha et al.
2003) and tannins, which are astringent and hemostatic (Cunha
et al. 2003).
Healing is a physiological process with the objective of repairing
damaged tissue. Synthetically, this mechanism has three stages
– inflammatory, proliferation and remodeling – that occur grad-
ually and dynamically (Mondolin and Bevilacqua 1985; Serhan
et al. 2008). Comfrey was cited as one of the most used plants
to heal wounds and to treat external skin problems, according to
the studies performed on the Brazilian population (Parente and
Rosa 2001; Luz 2001; Ritter et al. 2002; Champs et al. 2003;
Souza and Felfili 2006). More recently, different clinical trials
demonstrated the efficacy of comfrey extracts to treat sprains,
strains, muscle and joint problems (Predel et al. 2005; Kucera
et al. 2005; Staiger 2007), ankle distortions (Kucera et al. 2004;
Koll et al. 2004), and reducing acute back pain (Giannetti et al.
2010). To treat recent abrasions in patients, Symphytum herb
extract cream was used and it was a reduction of time to cica-
trize was observed a time reduction to cicatrize, with no adverse
effects (Barna et al. 2007).
The vehicle composition can affect drug release and skin
permeability properties, enhancing or reducing percutaneous
penetration. The selection of an appropriate vehicle is one of
the most important steps to increase the efficacy of a topically
applied bioactive (Kikwai et al. 2002; Nino et al. 2010).
In this way,the therapeutic efficacy of comfrey extracts in topical
formulations used to treat wounds could be dependent on the
composition and physicochemical properties of the vehicle.
In this context, the aim of this study was to evaluate the wound
healing ability of comfrey leave extract, incorporated in three
different pharmaceutical topical formulations. A commercial
extract was compared with a comfrey extract prepared in our lab-
oratory. For this purpose a wound healing rat model was used to
evaluate the effects on the microscopic aspects of wound lesions.
2. Investigations and results
2.1. Characterization of Symphytum officinale L. extracts
The physic-chemical parameters of the two extracts of S. offic-
inale L. were compared and both extracts were translucent,
Pharmazie 67 (2012) 355
ORIGINAL ARTICLES
Table 1: Physico-chemical characterization of Symphytum officinale L. extracts, CE and PE
Analysis Specifications*Commercial extract Prepared extract
Propyleneglycol concentration 35.0–50.0% 40 40
pH 4.00–8.50 6.20 6.90
Density (25 ◦C) 0.989–1.05 1.030 1.028
*Specifications provided by the manufacturer of the fluid glycolic comfrey extract
presenting characteristic smell and dark brown color. They are
also soluble in water, alcohol and propyleneglycol. The results
of propyleneglycol concentration, pH and density analysis are
shown in Table 1. The HPLC chromatograms highlighted the
same profile for both comfrey extracts. However allantoin is
peaking better in the extract prepared in the our laboratories
than in the commercial extract (Fig. 1).
2.2. In vivo evaluation of the wound healing process
2.2.1. Histological analysis
The presence of comfrey extract in formulations induced a more
effective wound healing process compared to the wounds not
treated, treated with excipients or treated with allantoin 5%.
The rate and intensity of the wound healing process, as well
as the structural organization of the tissue observed during the
treatment with comfrey were more favorable to induce a normal
pattern of the skin remodeling. In this context, among the formu-
lations studied, the O/W emulsion containing comfrey extract
3%, CE3%.E group induced a better and faster repair of dam-
aged tissue (Fig. 2). This can be identified by the presence of a
less intense inflammatory process and more collagen deposition
on the 3rd day (Fig. 2 C and L) compared to the control group
(C) (Fig. 2 A and J) and the group treated with allantoin (AE)
(Fig. 2 B and K). Moreover, on the 14th day the group treated
with comfrey emulsion (CE3%.E) presented a better resolution
of the inflammatory process, as well as better tissue organization
with a greater replacement of collagen fibers (Fig. 2 F and O).
On the 28th day it was observed that the treatment with com-
frey emulsion (CE3%.E) (Fig. 2 I and R) induced to a more
organized tissue, very close to a healthy skin. Based on these
findings, the O/W emulsion was selected as the pharmaceutical
Fig. 1: Comfrey extracts chromatograms obtained at 210 nm. Prepared extract (PE):
gray line; commercial extract (CE): black line. The arrow indicates the
allantoin peaks at the same retention time as internal standard
formulation to be used in order to compare the commercial and
prepared extracts of comfrey and the results are shown by the
morphometric analysis.
In accordance with qualitative analysis, the quantitative results
of morphometric analysis showed that the commercial comfrey
extract at 3% (CE3%) had a positive effect on the wound heal-
ing process induced by the three formulations studied, reducing
the amount of inflammatory cells present at the injury site and
promoting the collagen deposition.
This could be evidenced from day 3 to 28 by reduction of cellular
inflammatory infiltrate from 3% to 46% and increasing collagen
deposition from 40% to 240% (Table 2). Moreover, the O/W
emulsion containing comfrey (CE3%.E group) induced the best
wound healing process (Fig. 3 A1 and B1) compared to gel
(CE.G group) and solution (CE.S group), probably due to the
significant reduction of inflammatory cells number on the 21st
day (Fig. 3 A1).
Figure 3 (A2 and B2) shows the inflammatory process and
collagen deposition induced in the wounds treated with the
O/W emulsion containing commercial extracts at 3% (CE3%.E
group) and 8% (CE8%.E group) compared to the untreated
group (C group) and with the positive control group (allan-
toin at 5%, AE group). The higher concentration of commercial
comfrey extract (CE8%) in emulsion influenced positively the
wound healing process, reducing significantly the amount of
inflammatory cells on the 3rd, 14th and 21st days (Fig. 3 A2)
and increasing significantly the deposition of collagen on 7th
and 21st days (Fig. 3 B2). On the other hand, the results of mor-
phometric analysis of the wound healing process induced by
PE at 3% (PE3%.E group) and at 8% (PE8%.E group) (Fig. 3
Table 2: Percentages of reduction on cellular inflammatory
infiltrate and increasing in collagen deposition after
the treatment with O/W emulsion containing S. offic-
inale extracts or allantoin, compared with controls
groups
Groups Day 3 Day 7 Day 14 Day 21 Day 28
Cellular inflammatory infiltrate
Control 665.4 700.5 609.9 529.1 335.3
E−22,8% 4,2% −37,8% −33,0% −2,3%
CE3%.E −27,7% −3,9% −26,9% −42,1% −27,4%
CE8%.E −43,8% −21,5% −37,2% −41,8% −36,1%
PE3%.E −46,0% −20,4% −36,5% −36,1% −36,4%
PE8%.E −41,0% −24,9% −31,3% −45,1% −33,2%
AE −18,4% −17,8% −18,6% −40,8% −25,1%
Collagen deposition
Control 39503.0 90378.0 178265.0 179639.0 194672.0
E 31,8% 18,7% 30,0% 35,5% 34,4%
CE3%.E 98,5% 40,6% 42,8% 47,0% 58,4%
CE8%.E 134,3% 140,7% 52,0% 66,0% 36,6%
PE3%.E 156,5% 129,1% 71,7% 78,6% 71,0%
PE8%.E 244,0% 125,6% 90,2% 107,4% 110,0%
AE 44,2% 30,5% 14,1% 30,1% 29,6%
Data are expressed in percentage. Cellular nucleus and the collagen present in the skin fragments
were quantified in 20 randomly fields (total area covered equal to 1.5 ×106mm2)
356 Pharmazie 67 (2012)
ORIGINAL ARTICLES
Fig. 2: Photomicrographs of rat skin showing the structural organization rate and intensity of the wound healing process. C - Control (untreated); AE – O/W emulsion containing
allantoin 5%; CE3%.E - O/W emulsion containing CE 3%. Days: 3rd (A, B, C, J, K, L); 14th (D, E, F, M, N, O); 28th (G, H, I, P, Q, R). Hematoxylin-Eosin (A, B, C, D,
E, F, G, H, I) and Masson Trichrome (J, K, L, M, N, O, P, Q R), 200X
A3 and B3) are similar between them relative to the number
of inflammatory cellules, although PE at 8% induced the best
wound healing process in relationship to the deposition of col-
lagen, presenting a significative increase in all times evaluated
(Fig. 3 B3 and Fig. 4). Furthermore, PE at 8% (PE8%.E group)
is significantly better concerning the collagen deposition on the
28th days of analysis compared to CE at 8% (CE8%.E group).
The comparative analysis (Fig. 4) demonstrated that the wounds
treated with prepared comfrey emulsion at 8% (PE8%.E group)
showed a more efficient wound healing process, when compared
to the untreated group (C group), allantoin group (AE group) and
EC a 8% (CE8%.E group), characterized by the lower amount
of inflammatory cells (Fig. 4 A, C, E and G) and increase of
collagen deposition (Fig. 4 B, D, F and H) and this could be
Fig. 3: Inflammatory process (A1, A2, A3) and collagen deposition (B1, B2, B3) of rat skin. C – Control (untreated); CE3%.E – O/W emulsion containing CE3%; CE.G–
Carbomer gel containing CE 3%; CE.S – Glycero-alcoholic solution containing CE 3%; CE8%.E – O/W emulsion containing CE 8%; PE3%.E – O/W emulsion
containing PE 3%; PE8%.E – O/W emulsion containing PE 8%; AE – O/W emulsion containing allantoin 5%. Results are presented as means ±SEM. Statistically
significant data are given as *P< 0.05. Total area covered equal to 1.5×106mm2
Pharmazie 67 (2012) 357
ORIGINAL ARTICLES
Fig. 4: Photomicrographs of rat skin showing comparative analysis of wound healing process induced by extracts and allantoin. PE8%.E - O/W emulsion containing PE 8%;
CE8%.E - O/W emulsion containing CE 8%; AE - O/W emulsion containing 5% of allantoin; C - Control (untreated). Days: 3rd (A, B, C, D, E, F, G, H); 14th (I, J, K, L,
M, N, O, P); 28th (Q, R, S, T, U, V, X, Z). Hematoxylin-Eosin (A, C, E, G, I, K, M, O, Q, S, U, X) and Masson Trichrome (B, D, F, H, J, L, N, P, R, T, V, Z), 200X
observed from the 3rd day of treatment. Thus, on the 14th day
(Fig. 4 I, J, K, L, M, N, O and P) it was possible to observe
for PE8%.E group a more organized scar tissue (Fig. 4 A, B,
I, J, Q and R), and on the 28th day (Fig. 4 Q and R) the scar
tissue was very close to the normal tissue, presenting a great
tissue organization and presence of skin appendages, indicating
the better efficacy of this treatment.
3. Discussion
The data of the physico-chemical parameters suggest that both
extracts were in accordance to the pharmacopeical specifica-
tions. The HPLC chromatogram (Fig. 1) indicates a similarity of
chemical composition profile, however with marked differences
in number and intensity of some peaks. Allantoin, reported to
be one of the main compounds responsible for the wound heal-
ing properties of the comfrey extract (Saito and Oliveira 1986;
Cunha et al. 2003; Carvalho 2004) was found only in the PE
extract.
Wound healing is a physical, chemical and biological process
which starts immediately after a tissue injury in order to repair
the damaged tissue. It is a complex phenomenon and of great
importance in skin regeneration but, unfortunately, still unclear
in detail (Sanchez Neto et al. 1993).
In the present work, leave extracts of S. officinale L. presented
wound healing activity, thereby justifying their extensive use in
Brazil folk medicine, as already observed by different authors
(Parente and Rosa 2001; Luz 2001; Ritter et al. 2002; Champs
et al. 2003; Souza and Felfili 2006) and in clinical studies con-
ducted in other countries (Kucera et al. 2000; Koll et al. 2004;
Kucera et al. 2004, Predel et al. 2005; Kucera et al. 2005; Staiger
2007; Giannetti et al. 2010). The inflammatory response is an
important step of the wound healing process as it prepares the
environment of the wound repair itself. However, this stage
should not be extremely intense, because an excessive inflam-
matory response could cause delay in wound healing favoring
the disturbance between synthesis and degradation of colla-
gen, and promoting degradation of the matrix (Ashcroft et al.
2002). Our results suggest that the comfrey extract modulates
the inflammatory response possibly by inhibiting the chemotaxis
of inflammatory cells to the site of the wound, thus prevent-
ing the release of reactive species responsible for the oxidative
stress and tissue damage, as described by Bradbury et al. (1993)
in a study of pathogenesis of vascular diseases. Moreover, the
well formed collagen fibers observed in PE3%.E and PE8%.E
(prepared comfrey extract at 3% and 8%, respectively) support
the effectiveness of comfrey in fibroblastic proliferation and
synthesis of extracellular matrix during wound healing.
Allantoin is widely used in pharmaceutical and cosmetics prepa-
rations due to its wound healing properties as anti-irritant,
moisturizer and necrotic tissue remover (Saito and Oliveira
1986; Oliveira et al. 2008). Due to these related properties,
allantoin was use as a positive control in this work. The results
obtained here showed that allantoin at 5% in soft lotion O/W
emulsion has a wound healing effect when compared with the
controls groups, however its activity was lower compared to the
extracts. Thus, the differences in healing effects observed in this
work between PE and CE at 8% should not be attributed only to
allantoin, better evidenced in PE extract (Fig. 1). Araújo et al.
(2010) showed, for the first time, the histological wound healing
profile induced by allantoin in rats and demonstrated that it is
able to ameliorate and fasten the reestablishment of the normal
skin.
From Table 2 it can be observed that healing estimated as col-
lagen synthesis is dose dependent and it was maintained up
to complete healing. It is also dependent of the extract prepa-
ration method, different collection location or probably from
seasonal influences of comfrey leave collection. It can be evi-
denced that PE has a stronger effect in both parameters of healing
during the experimental period of observation. Cell migration
to wound tissue was increased especially in days 7–14 post
injury. At the same days stronger effects of S. officinale extracts
were also observed, suggesting modulation of the inflamma-
tory processes. These results about the wound healing activity
of comfrey extract related to its anti-inflammatory effect are in
agreement with the clinical studies results described by Kucera
et al. (2000); Koll et al. (2004), Kucera et al. (2004), Kucera et
al. (2005), Barna et al. (2007).
In particular, the results obtained in this work corroborate with
the clinical study of Barna et al. (2007), that also used a topi-
cally applied preparation (Traumaplant®) containing 10% active
ingredient from the aerial parts of medicinal comfrey (Sym-
phytum ×uplandicum NYMAN). Furthermore, in this study the
patients did not reported adverse reactions or tolerability prob-
lems, thus indicating the safety of the topical use of aerial parts
extracts.
Aditionally, our results confirm the anti-inflammatory properties
of the topical comfrey products described by Kucera et al. (2004)
in a randomized double blind study in patients with acute ankle
distortions.
The present work demonstrates that wound healing processes
can be induced by comfrey extract, which modulates the inflam-
matory process and stimulates the production of collagen,
evidenced by morphometric analysis. Furthermore, the O/W
emulsion showed to be the vehicle most effective to induce heal-
ing activity, particularly with prepared extract obtained from
358 Pharmazie 67 (2012)
ORIGINAL ARTICLES
comfrey leaves collected in Minas Gerais state in Brazil. It
shows the best efficacy to control the inflammatory process and
to induce collagen deposition at 8% concentration.
It is clear that the higher concentration of Symphytum active
ingredients in the PE8%.E was responsible for the best control of
the inflammatory process and collagen deposition. These results
are in agreement with the results found by Kucera et al. (2004;
2005), where the cream with the highest concentration of extract
showed better results.
4. Experimental
4.1. Plant material
Leaves of Symphytum officinale L. were collected in Betim, Minas Gerais,
Brazil, in April 2007. The voucher specimen (OUPR 21806) was identi-
fied and deposited at the Herbarium of the Instituto de Ciências Exatas e
Biológicas, Universidade Federal de Ouro Preto (UFOP), Ouro Preto, Minas
Gerais, Brazil.
4.2. Preparation of extract
Leaves were dried at 37 ◦C for two days, reduced to powder (255.0 g) and
extracted by maceration with a mixture of water and propyleneglycol (60:40)
for 48 h before filtration (Brasil 1959). This extract was prepared at the same
concentration of the commercial glycolic extract.
A commercial glycolic extract from leaves of S. officinale L. containing 20%
w/v from dry plant was obtained from a Brazilian manufacturer (Natural
Pharma® Brazil).
The reagents used were analytical grade and the MilliQ water was purified
by the Symplicity 185 System (Millipore®, Brazil). Allantoin was purchased
from Sigma-Aldrich®(Brazil). The reagents used were analytical grade and
the MilliQ water was purified by the Symplicity 185 System (Millipore®,
Brazil).
4.3. Characterization of the extracts
The extracts were evaluated considering physical appearance, smell, color,
pH (pH 300M Analyzer, Brazil) and solubility. The chromatographic pro-
files were obtained on a HPLC system Waters2695 Alliance System (Waters
Corporation, Milford, EUA) using a 2996 PhotoDiodo Array Detector. The
chromatographic profiles of extracts were achieved by using an ODS2
column (Spherisorb Waters 4.6 ×150 mm, 5 mm) and Phenomenex guard
column (C18 4.0 ×3.0 mm). The mobile phase were: solvent A, HPLC grade
acetonitrile (Tedia, Brazil) filtered using 0.45 mm Millipore®membrane and
solvent B, MilliQ water. The gradient profile was: 0, 5, 10, 15 and 20min,
A%: 1, 1, 100, 100 and 1, respectively. Chromatograms were obtained at
25 ◦C at 210 nm wavelength. Eluted mobile phase was monitored at 200 –
800 nm. The flow rate was 1 ml/min and the injection volume was 50 ml of
diluted extract samples (extract/water, 1:1), which were filtered through a
0.20 mm filter (Syringe Filter, Nalgene).
4.4. Preparation of pharmaceutical formulations
Three formulations containing Symphytum officinale L extract were
evaluated: carbomer®gel (1.5 g carbopol®940, 10 ml glycerin, 0.5 ml tri-
ethanolamine, 0.15 g methylparaben, 0.10 g propylparaben, 87.75 ml water)
glycero-alcoholic solution (40 ml glycerin, 1 ml dimethylsulfoxide, 59 ml
alcohol 60 ◦GL) and a oil/water (O/W) emulsion (soft lotion) (3.6 g
cetylestearyl alcohol, 0.4 g sodium cetylestearyl sulfate, 4 ml mineral oil,
5 ml sorbitol solution 70% w/v, 0.15 g methylparaben, 0.10 g propylparaben,
86.75 ml water). The comfrey commercial glycolic extract (CE) at 3%
w/w was incorporated. After this first evaluation the two comfrey glycolic
extracts, commercial (CE) and prepared (PE), were incorporated into emul-
sion, at 3 and/or 8% w/w. A positive control with allantoin 5% in the same
preparation was used for comparison.
4.5. Animals
Female Wistar rats (180–200 g) were used and housed individually, on a
12 hours light/dark cycle with a standard pellet diet (Labcil Petilizado-Socil,
Brazil) and water ad libitum. The experimental protocol was approved by
the Ethical Committee of UFOP (number 2007/98) and was in accordance
to the Guide for the Care and Use of Laboratory Animals, published by the
US National Institute of Health (NIH Publication, revised in 1985).
4.6. Wound healing evaluation
The animals were anesthetized by intraperitoneal with sodium pentobarbital
(50 mg/kg). The hair of the dorsal back of each animal was removed and
an excision wound (1 cm2) was made by removing a full thickness piece of
the skin (Sekine et al. 1998). The animals were randomly distributed in 13
groups (n = 4): control untreated (C), carbomer gel excipients (G), carbomer
gel with CE 3% (CE.G), carbomer gel with allantoin 5% (AG); glycero-
alcoholic solution excipients (S); glycero-alcoholic solution with CE 3%
(CE.S), glycero-alcoholic solution with allantoin 5% (AS); O/W emulsion
excipients (E); O/W emulsion with CE 3% (CE3%.E); O/W emulsion with
CE 8% (CE8%.E); O/W emulsion with PE 3% (PE3%.E); O/W emulsion
with PE 8% (PE8%.E) and O/W emulsion with allantoin 5% (AE). The
weighed topical formulations (0.25 g) were administered daily during 14
days (Goldman et al. 1985).
4.7. Histopathological analysis
After sacrifice (sodium pentobarbital, 100 mg/kg) the wounds of animals
were excised on the 3rd, 7th , 14th, 21st and 28th days after the surgery,
containing a margin of normal skin around the wound. The tissues were pre-
served in 10% buffered formalin. Fourmm thickness sections were stained
with hematoxylin-eosin and Masson Trichrome (Bec¸ak and Paulete 1976;
Behmer et al. 1976). For the qualitative analysis, the specimens were
assessed under a light microscope (Olympus CH30, Japan) in order to ana-
lyze the new epithelium, inflammation, vascular responses and the collagen
formation.
The quantitative analysis (morphometry) was performed using scans of the
tissues for determination of intensity of the inflammation and collagen
deposition. Cellular nucleus and the collagen present in the skin frag-
ments were quantified in 20 randomly fields (total area covered equal to
1.5 ×106mm2). The images were amplified, acquired by a Microcamera
Leica and the programe DM5000B Leica Application Suite (Version 2.4.0
R1 Leica Microsystems, Switzerland Ltd) and analyzed by Leica programe
QWin V3 (Leica Microsystems, Switzerland Ltd). The tissues were ana-
lyzed microscopically,qualitative and quantitatively,by the same pathologist
without prior knowledge of the identity of the groups.
4.8. Statistical analysis
The results of stability were expressed as means ±SD, and statistical eval-
uation was performed by Kruskal-Wallis and Dunns post-test. The data of
histological analysis were expressed as means ±SEM, and statistical eval-
uation was performed by ANOVA and Tukey post-test. Values lower than
P< 0.05 were considered significant.
Acknowledgments: This work received the financial support of Fundac¸ão de
Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG- APQ-6247–07
and Redes TOXIFAR and NANOBIOMG. We are grateful to the scholarship
afforded by Universidade Federal de Ouro Preto (UFOP) to the first author;
to the Salão do Encontro, Betim, MG, Brazil, for the donation of the plant;
to the Dr. Ilza Damázio for the technical support for the HPLC analysis and
to Prof. Dr. Viviane Scalon for the botanical classification of Symphytum
officinale L.
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