Phenolic content, antioxidant activity and effect on collagen synthesis of a traditional wound healing polyherbal formula

Abstract

The aim of this study was to investigate the ethanol extracts of four medicinal plants, Achillea millefolium L., Hyssopus officinalis L., Equisetum arvense L. and Echinacea purpurea L. and their polyherbal formula, used in traditional medicine for wound healing. The study analyzed their total phenolics content using Folin-Ciocalteu method and identified the main constituents by HPLC. Their antioxidant activity was evaluated by DPPH and ABTS assays and the formula’s capacity to enhance collagen synthesis in L929 fibroblast cell culture was determined by Sircol assay. The results showed that the polyherbal extract had phenolic constituents with pharmacological properties: chlorogenic acid, caffeic acid, luteolin and apigenin. It was showed that the polyherbal formula presented higher antioxidant activity than plant extracts and induced a stimulation of collagen synthesis by fibroblasts, which could contribute to wound strength. In conclusion, the proposed polyherbal formula demonstrated high potential as therapeutic agent in wound healing.

Full text

Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele Vieţii
Vol. 25, issue 1, 2015, pp.41-46
© 2015 Vasile Goldis University Press (www.studiauniversitatis.ro)
*Correspondence: Dr. Valentina Alexandru, National Institute of R&D for Biological Sciences, 296, Splaiul Independentei, sector 6,
060031, Bucharest, Romania, Tel/fax: +40-21-2200882, E-mail: valentinaalexandru@yahoo.com
Article published: February 2015
PHENOLIC CONTENT, ANTIOXIDANT ACTIVITY AND EFFECT ON
COLLAGEN SYNTHESIS OF A TRADITIONAL WOUND HEALING
POLYHERBAL FORMULA
Valentina Alexandru1*, Alexandra Gaspar1, Simona Savin1, Agnes Toma1,
Rodica Tatia1, Elvira Gille2
1National Institute of R&D for Biological Sciences, 296, Splaiul Independentei, sector 6, 060031,
Bucharest, Romania
2National Institute of R&D for Biological Sciences, „Stejarul” Biological Research Centre, 6,
Alexandru cel Bun Street, 610004, Piatra Neamt, Romania
ABSTRACT. The aim of this study was to investigate the ethanol extracts of four medicinal plants, Achillea
millefolium L., Hyssopus officinalis L., Equisetum arvense L. and Echinacea purpurea L. and their polyherbal
formula, used in traditional medicine for wound healing. The study analyzed their total phenolics content
using Folin-Ciocalteu method and identified the main constituents by HPLC. Their antioxidant activity was
evaluated by DPPH and ABTS assays and the formula’s capacity to enhance collagen synthesis in L929
fibroblast cell culture was determined by Sircol assay. The results showed that the polyherbal extract had
phenolic constituents with pharmacological properties: chlorogenic acid, caffeic acid, luteolin and apigenin. It
was showed that the polyherbal formula presented higher antioxidant activity than plant extracts and induced
a stimulation of collagen synthesis by fibroblasts, which could contribute to wound strength. In conclusion,
the proposed polyherbal formula demonstrated high potential as therapeutic agent in wound healing.
Keywords: polyherbal formula, wound healing, antioxidant activity, collagen, medicinal plants
INTRODUCTION:
Oxidative stress is caused by an imbalance between
the production of reactive oxygen species (ROS) and
the endogenous antioxidant system. The human body
cells are equipped with multiple mechanisms to fight
against ROS and to maintain the cellular redox
homeostasis (Bergendi et al., 1999). When the
antioxidant protection mechanism became unbalanced,
the exogenous antioxidants, such as those from plants,
can help reducing the oxidative damage. The phenolic
compounds (phenolic acids, flavonoids, flavanols,
anthocyanins, etc.) from medicinal plants have been
reported to be potent free radical scavengers (Mathew
et al., 2006). The antioxidant properties of phenolic
compounds have been substantiated by their high
reactivity and potential to chelate metal ions (Rice-
Evans et al., 1997).
In acute and chronic wounds, the expression of
enzymatic antioxidants increased, while their activity
decreased, due to high oxidative stress (James et al.,
2001). Besides, several studies reported that depletion
of non-enzymatic antioxidants was more pronounced in
chronic wounds than in acute wounds (Shukla et al.,
1999; Steiling et al., 1999). Addition of substances
with antioxidant effect was proved to be important in
the successful treatment of skin wounds (Houghton et
al., 2005).
Healing of wounds involves the activity of an
intricate network of blood cells, cytokines and growth
factors, resulting in restoration of normal skin tissue
condition (Clark, 1991). The interest in evaluating the
utility of plant extracts for wound healing has been
increased during the last decade. The importance of
plant secondary metabolites as potential agents that
interfered with various wound repair stages has been
demonstrated, both in vitro and in vivo (Parasanta et
al., 2013; Tsala et al., 2013).
Traditional medicine often used multiple herb
formulae for a wide range of treatments. In skin wound
healing, four medicinal herbs, Achillea millefolium L.
(Compositae), Hyssopus officinalis L. (Labiatae),
Equisetum arvense L. (Equisetaceae) and Echinacea
purpurea L. (Compositae) were used either alone or in
combination with other herbs. These plants contributed
to wound healing and tissue regeneration by multiple
mechanisms, which still need assessment and
validation by scientific studies.
The present study aimed to evaluate, for the first
time, their combination in a particular polyherbal
formula. Its assessment consisted of the identification
and quantification of polyphenolic compounds and the
determination of total antioxidant activity. In order to
support the use of this four-herb formula as a new,
natural product for skin wound healing, it was also
investigated its in vitro effect on collagen secretion by
L929 fibroblast cells in culture.
MATERIALS AND METHODS:
Materials
The plants Equisetum arvense L., Achillea
millefolium L., Hyssopus officinalis L and Echinacea
purpurea L. were collected from Neamt and Suceava
counties, located in the North of Romania. The plant
material was authenticated by prof. dr. Nicolae Stefan
(Botany Department, Faculty of Biology, “Alexandru
Ioan Cuza” University, Iasi). Voucher specimens were
deposited at the Herbarium of Iasi Botanical Garden,
Romania. HPLC-grade gallic acid, chlorogenic acid,
caffeic acid, coumaric acid, ferulic acid, rutoside,
myricetin, luteolin, quercetin, apigenin, acetonitrile and
methanol were purchased from Sigma-Aldrich

Alexandru V., Gaspar A., Savin S., Toma A., Tatia R., Gille E.
Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele Vieţii
Vol. 25, issue 1, 2015, pp. 41-46
© 2015 Vasile Goldis University Press (www.studiauniversitatis.ro)
42
(Germany). Butylated hydroxytoluene (BHT), Folin-
Ciocalteu’s phenol reagent, 6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid (Trolox), 2,2-
diphenyl-1-picryl-hydrazyl (DPPH) and 2,2’-
azinobis(3-ethylbenzothiazoline-6-sulfonic acid)
diammonium salt (ABTS) and all other chemicals and
solvents of analytical grade were purchased from
Sigma-Aldrich (Germany). The fibroblast cell line
NCTC clone L-929 was purchased from the European
Collection of Cell Cultures (ECACC), minimum
essential medium Eagle (MEM) from Sigma-Aldrich
(Germany) and fetal calf serum (FCS) from Biochrom
AG (Germany). Sircol collagen assay kit was
purchased from Biocolor Ltd. (Newtownabbey, UK).
Extraction procedures
The aerial parts of each plant were air dried, in the
dark and minced using a blender. In order to obtain the
polyherbal extract, dried herbs were mixed as follows:
4 g Equisetum arvense, 3 g Achillea millefolium, 2.5 g
Echinacea purpurea, 0.5 g Hyssopus officinalis. The
mixture (10 g) was extracted in 100 mL ethanol (70 %,
v/v), at room temperature, in the dark, for 10 days.
Then, the polyherbal extract was separated from the
residue by filtration through Whatman No.1 filter paper
and concentrated under vacuum, at 40 °C using a rotary
evaporator (VVMicro, Heidolph, Germany. For cell
culture experiments, the solid residue of the polyherbal
extract, resulted after concentration under vacuum, was
weighed, dissolved in distilled water and sterilized by
filtration through 0.2 µm membrane. On the
experiment day, several extract dilutions were prepared
in the culture medium. Individual plant ethanol extracts
were prepared in the same conditions, in order to be
used as controls.
Total phenolics content assay
Total phenolics content of the herb extracts was
determined using a modified Folin-Ciocalteu method
(Singleton et al., 1999). Briefly, 2.5 mL herb extract
was mixed with 2.5 mL Folin-Ciocalteu reagent and,
after 5 min, 2 mL sodium carbonate (12%, w/w) were
added. The mixture was allowed to stand at room
temperature, for 15 min. The optical density (OD) of
the resulting blue complex was measured at 731 nm
using an UV-Vis spectrophotometer (Jasco V-650,
Japan). Total phenolic content was calculated from the
linear equation of the calibration curve obtained for
chlorogenic acid. The results were expressed as mg
chlorogenic acid equivalents (ChAE)/g dry extract.
DPPH free radical scavenging activity assay
The method is based on scavenging DPPH stable
radical in the presence of hydrogen donor antioxidant,
along with color turn from purple to yellow. We
measured the free radical scavenging activity of each
extract using the method of Hatano et al. (1988) with
some modifications. Briefly, different herb extract
concentrations (10, 25, 50, 100, 250, 500 µg/mL) were
added to DPPH methanol solution (0.25 mM) and each
mixture was incubated in the dark, for 30 min. The OD
was measured at 517 nm against the blank (DPPH
methanol solution), using an UV/VIS
spectrophotometer (Jasco V650, Japan). The inhibition
percentage was calculated using the following formula:
Inhibition (%) = (ODblank-ODsample) / ODblank x100 (1)
The sample concentration that inhibited 50% of
DPPH free radicals (IC50, µg/mL) was calculated from
the graph plotting inhibition percentage against extract
concentration by linear regression analysis. BHT was
used as positive control.
ABTS radical cation scavenging assay
The method is based on the capacity of a sample to
scavenge the ABTS radical cation (ABTS•+), compared
to Trolox as standard antioxidant. We determined the
antioxidant activity of each extract according to the
method of Rice-Evans and Miller (1994). Briefly, 2.5
mL ABTS stock solution (7 mM) in potassium
persulfate (2.45 mM) was mixed with 0.1 mL sample
(herb extract) or standard (Trolox) and 0.4 mL ethanol,
and the mixture was allowed to stand at room
temperature, for 3 min. Then, the OD was recorded at
731 nm against the blank, containing all reagents
except the tested extract, at an UV/VIS
spectrophotometer (Jasco V 650). The results were
expressed as Trolox equivalents antioxidant capacity
(TEAC) calculated using the formula:
TEAC (μM Trolox equivalents/g dry weight) = CTrolox x
f x (ODsample – ODblank) / (ODTrolox – ODblank) (2)
where Ctrolox is Trolox concentration and f is the
sample dilution factor.
HPLC analysis
The separation, identification and quantification of
phenolic compounds from polyherbal extract were
performed by HPLC, using an Agilent 1200 system
(Agilent, USA) equipped with diode array detector and
Eclipse XDB-C18 (150 x 4.6 mm i.d.; 5 µm particles)
chromatographic column, after injection of 10 µl
sample. The mobile phase used for phenolics
separation was a mixture of phase A (2 mM sodium
acetate buffer, pH 3.05) and phase B (acetonitrile), in
linear gradient mode, as follows: 0-30 min, 2-20% B in
A; 30-40 min, 20-30% B in A; 40-50 min, 30% B in A;
50-60 min, 30%-2% B in A. The flow rate was 1
mL/min. Chromatograms were recorded at a
wavelength of 260 nm for phenolic acids and 320 nm
for flavonoids. The constituents present in the
polyherbal extract were identified by comparing the
recorded UV profile and their retention times with
those obtained for a mixture of known standards of
phenolic acids (gallic acid, chlorogenic acid, caffeic
acid, p-coumaric acid, ferulic acid) and flavonoids
(rutoside, myricetin, luteolin, quercetin, apigenin). In
order to calculate the content of each polyphenol
identified in the polyherbal extract, calibration curves
for standards were built as five-point plots, in the range
of 0.976 – 15.625 µg/mL.
Soluble collagen assay
Mouse fibroblasts cell culture (NCTC cell line
clone L929) was used to study the effect of polyherbal
extract on collagen secretion. Cells were seeded in the
wells of 24-well culture plate, at a density of 5x104
cells/well in MEM supplemented with10% FCS.

Phenolic content, antioxidant activity and effect on collagen synthesis of a traditional wound healing polyherbal formula
Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele Vieţii
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43
After 24 h of incubation in standard conditions, the
cells adhered to plastic and the culture medium was
changed with MEM supplemented with 5% FCS,
containing different concentrations of polyherbal
extract (35-140 µg/mL). The plates were incubated at
37 ºC, in humidified 5% CO2 air atmosphere, for 48 h
and 72 h, respectively. The control group consisted of
untreated cells cultivated in MEM with 5% FCS.
Collagen secretion in the culture medium was
determined using Sircol collagen assay kit, according
to manufacturer’s instructions. Briefly, the harvested
culture media were centrifuged at 1,500 rpm, for 4 min
and, then, 100 μl supernatant was mixed with 1 mL
Sircol dye, for 30 min. The mixture was centrifuged at
10,000 rpm, for 5 min to precipitate the collagen-dye
complex. Then, the pellets were dissolved in 1 mL
alkali reagent and vortexed. The OD of the solution
was read at 540 nm using Sunrise microplate reader
(Tecan, Austria).
Statistical analysis
All chemical analyses were run in triplicate and
three cell culture independent experiments were
performed in three replicates. Data were reported as
mean ± standard deviation (SD). Pair comparison of
control and each sample was carried out by t-test.
Significant statistical differences were considered at p
< 0.05.
RESULTS AND DISCUSSIONS
Total phenolics content and antioxidant
activity
The results of total phenolics content in E. arvense,
H. officinalis, A. millefolium, E. purpurea ethanolic
extracts and their polyherbal formula extract are
showed in Fig. 1. The amount of total phenolics in
plant extracts varied from 8.95 mg ChAE/g dry extract
for E. purpurea, to 12.43 mg ChAE/g dry extract for A.
millefolium. Significant (p < 0.05) higher phenolic
compounds level was detected in the polyherbal extract
(14.42 mg ChAE/g dry extract), compared to each
plant extract.
Fig. 1 Total phenolics content in plant extracts and polyherbal (PH) extract. *p < 0.05, compared to each plant extract
In order to determine the antioxidant activity of the
polyherbal extract, in comparison to individual plant
extracts, two complementary test systems have been
applied, DPPH and ABTS assays. The results of DPPH
assay showed the extract concentration that resulted in
50% DPPH free radical inhibition (IC50) (Fig. 2).
Fig. 2 Radical scavenging activities of plant extracts and polyherbal (PH) extract on DPPH radical. Results were
expressed as IC50 (mean ± SD). BHT was used as standard reference. *p<0.05, compared to BHT; #p<0.05, compared
to plant extracts.

Alexandru V., Gaspar A., Savin S., Toma A., Tatia R., Gille E.
Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele Vieţii
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44
IC50 values decreased in the folowing order: H.
officinalis >E. arvense >E. purpurea >A. millefolium >
polyherbal extract. Therefore, the polyherbal extract
presented a significant (p < 0.05) higher radical
scavenging activity than individual plant extracts, but
significant (p < 0.05) lower than BHT, a well-known
synthetic antioxidant.
The antioxidant activities of plant extracts
evaluated by ABTS assay varied from 98.13 µM
Trolox equivalents/g dry extract for E. arvense to
176.19 µM Trolox equivalents/g dry extract for A.
millefolium (Table 1). The polyherbal extract had the
highest antioxidant activity (254.88 μM Trolox
equivalents/g dry extract). This result was in
accordance with that obtained by DPPH assay and
correlated with its phenolic compounds content.
Table 1.
Antioxidant activities of plant extracts and polyherbal formula extract evaluated by ABTS assay
Plant species
ABTS assay
(μM Trolox equivalents/g dry weight)
Hyssopus officinalis
171.73 ± 4.54
Echinacea purpurea
168.22 ± 8.48
Achillea millefolium
176.19 ± 4.96
Equisetum arvense
98.13 ± 3.84
Polyherbal extract
254.88 ± 12.67*
*p < 0.05, compared to each plant extract
All these data showed that the polyherbal extract
exhibited higher phenolics content and antioxidant
capacity, compared to its component extracts of A.
millefolium, E. purpurea, E. arvense and H. officinalis.
As a result, the polyherbal extract was tested in
subsequent analyzes.
Chemical composition of the polyherbal
extract
The established HPLC method was applied as
analytic approach to determine the major compounds
of the polyherbal extract. The recorded HPLC profile
presented nine main peaks, at 1.312, 6.352, 18.616,
21.899, 24.819, 25.169, 29.190, 29.541 and 45.067 min
(Fig. 3).
Fig. 3 Chromatographic profile of polyphenolic constituents from the polyherbal extract recorded by HPLC
A mixture of known pure compounds was also
chromatographed and used as external standards of
phenolic acids (gallic acid, chlorogenic acid, caffeic
acid, coumaric acid and ferulic acid) and flavonoids
(rutoside, myricetin, luteolin, quercetin and apigenin).
The values of retention time for these standards and
their calibration curves parameters are presented in
Table 2. Table 2.
Analytical results of calibration curves of ten polyphenolic compounds used as standards in HPLC analysis
Standard
Retention time
(min)
Regression equation of
the calibration curvea
Correlation factor
R2
Gallic acid
4.451
y = 28.963x + 41.860
0.985
Chlorogenic acid
14.485
y = 14.856x + 19.038
0.988
Caffeic acid
17.440
y = 24.325x + 39.374
0.984
Coumaric acid
23.115
y = 19.319x + 33.477
0.982
Ferulic acid
26.018
y = 24.906x + 42.230
0.982
Rutoside
28.305
y = 14.240x + 20.226
0.987
Myricetin
35.800
y = 33.706x + 99.057
0.903
Luteolin
44.904
y = 58.234x + 179.806
0.920
Quercetin
45.302
y = 45.482x + 139.321
0.914
Apigenin
53.747
y = 33.958x + 121.724
0.908
aThe calibration curves were plotted in linear regression analysis of the integrated peak area (y) versus concentration (x)

Phenolic content, antioxidant activity and effect on collagen synthesis of a traditional wound healing polyherbal formula
Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele Vieţii
Vol. 25, issue 1, 2015, pp. 41-46
© 2015 Vasile Goldis University Press (www.studiauniversitatis.ro)
45
In order to determine the content of the identified
compounds in polyherbal extract, quantitative
calculations were performed by peak area integration.
The results of HPLC analysis showed that the
polyherbal extract presented high levels of chlorogenic
acid (1.226 mg/g dry extract), rutoside (1.605 mg/g dry
extract), apigenin (0.982 mg/g dry extract) and luteolin
(0.692 mg/g dry extract) (Table 3). Low levels of
caffeic acid, coumaric acid and quercetin were
quantified in the polyherbal extract (Table 3).
Table 3.
Content of phenolic acid and flavonoid compounds
identified in the polyherbal extract
Compound
Content in the polyherbal extract
(mg/g dry weight)
Gallic acid
ND
Chlorogenic acid
1.226 ± 0.025
Caffeic acid
0.252 ± 0.050
Coumaric acid
0.251 ± 0.092
Ferulic acid
0.014 ± 0.003
Rutoside
1.605 ± 0.224
Myricetin
ND
Luteolin
0.692 ± 0.148
Quercetin
0.081 ± 0.008
Apigenin
0.982 ± 0.281
ND - not detected
Previous studies showed that these phenolic
compounds presented several pharmacological
properties and exerted anti-inflammatory, antioxidant,
antiviral, antibacterial and vulnerary activities (Fuchs
et al., 1993; Morishita et al., 2001; Song et al., 2008;
Lopez-Lazaro, 2009; Kostyuk et al., 2010).
Effect of the polyherbal extract on collagen
secretion
Wound healing is a fundamental response to tissue
injury. The present knowledge described three phases
of this process: inflammatory phase, proliferative phase
and remodelling phase. In the proliferative phase,
fibroblasts produced a variety of substances, essential
for wound repair, including glycosaminoglycans and
collagen (Madden et al., 1968). In the remodeling
phase, new collagen was formed and tissue tensile
strength was increased due to intermolecular cross-
linking of collagen, via vitamin C-dependent
hydroxylation (Prockop et al., 1979; Stadelmann et al.,
1998). In Fig. 4 is presented the influence of polyherbal
extract on the synthesis of soluble collagen, after
cultivation in different concentrations with L929
fibroblast cells.
Fig. 4 Determination of collagen secretion by L929 fibroblast cells incubated with different concentrations of polyherbal
extract, for 48 h and 72 h, using Sircol assay. Three independent experiments were performed with three replicates for
each sample. Values are mean ± SD. *p < 0.05, compared to control (untreated cells).
The results showed a significantly (p < 0.05)
increase of collagen synthesis in the culture medium of
fibroblasts treated with 70 and 140 µg/mL polyherbal
extract, after 48 h and 72 h of cultivation. It was
observed that the collagen synthesis was almost 2 times
higher in cultures treated with 140 µg/mL polyherbal
extract, for 72 h, compared to the value obtained in the
control group (0 µg/mL polyherbal extract).
Previous studies reported that natural polyphenols
presented reducing properties, protection of
intracellular lipids from oxidation and influenced
collagen synthesis (Mucha et al., 2013). Our
experimental data suggested that increased collagen
synthesis in L929 fibroblast cell culture was correlated
to high phenolics content and, by default, with the
antioxidant activity of the polyherbal extract.
CONCLUSIONS:
These results support the traditional use of this
four-herb formula for wound care. The polyherbal
extract had higher total phenolics content and
antioxidant activity, compared to individual plant
extracts. It was showed its in vitro capacity to stimulate
collagen synthesis in a culture of fibroblast cells.
Therefore, this combination of plant extracts may be
useful as therapeutic agent in wound healing. Future
studies could be performed, in order to find out its
unexplored efficacy and high potential as a source of
natural health care products.

Alexandru V., Gaspar A., Savin S., Toma A., Tatia R., Gille E.
Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele Vieţii
Vol. 25, issue 1, 2015, pp. 41-46
© 2015 Vasile Goldis University Press (www.studiauniversitatis.ro)
46
ACKNOWLEDGMENT:
This study was supported by the Executive Unit for
Financing Higher Education, Research, Development
and Innovation (UEFISCDI), Project No. 62071 and
Romanian Project BIODIV No. 102.
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