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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.
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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
Vol. 25, issue 1, 2015, pp. 41-46
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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
Gallic acid
Chlorogenic acid
Caffeic acid
Coumaric acid
Ferulic acid
Rutoside
Myricetin
Luteolin
Quercetin
Apigenin
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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Phytopharmacol, 4, 532-560, 2013.
... Kaempferol 3-O-sophoroside and kaempferol 3-O-sophoroside-7-O-glucoside, isolated from E. giganteum , are reported to be used as potent pharmacological agents in treating gastrointestinal diseases, oxidative stress, and histologic changes (Campos-Vidal et al., 2022). Apigenin 5-O-glucoside, isolated from E. arvense (Zhang et al., 2015), was revealed as an effective Caffeic acid, p-coumaric acid (Uslu et al., 2013;Alexandru et al., 2015); vanillic acid (Uslu et al., 2013); ferulic acid ( (Syrchina et al., 1980) ...
... Benzoic acid, isolated from the stems of E. arvense, E. fluviatile, and E. telmateia (Hiraga et al., 1997), is a commonly used preservative in acidic foods, beverages and also a widely used compound in pharmaceutical industry (Zu et al., 2017). Gallic acid, chlorogenic acid, myricetin (Alexandru et al., 2015), trans-caffeoyl-3-β-glucoside (Park and Tomohiko, 2011), (6R,7aS)-5,6,7,7a-tetrahydro-6-hydroxy-4, 4,7a-trimethyl benzofuran-2(4H)-one , and a phenylpropanoid, rosmarinic acid (Alves et al., 2016) were isolated from the aerial parts of E. hyemale and these compounds have antioxidant, anti-inflammatory, anticancer, antidiabetic, osteoporosis protection, gastrointestinal, neuropsychological, cardiovascular disorders, anti-obesity, hepatoprotective, and antineoplastic properties (Naveed et al., 2018;Kahkeshani et al., 2019;Imran et al., 2021;Guan et al., 2022). ...
... Methanolic extracts of the whole plant parts of E. arvense showed 90% DPPH inhibition, and 70% nitric oxide inhibition and 60% H 2 O 2 inhibition (Amit et al., 2013). Similarly, a number of studies have found that the whole plant parts and aerial parts of E. arvense have significant antioxidant effects (Jia-Gua et al., 2006;Stajner et al., 2006Stajner et al., , 2009Oka et al., 2007;Milutinovic et al., 2014;Cetojevic-Simin et al., 2010;Uslu et al., 2013;Alexandru et al., 2015;Huh and Han, 2015). Aqueous extracts and fractions of E. arvense aerial parts showed notable DPPH and hydroxyl free radical scavenging effects with an EC 50 of 1.52 ± 0.07 mg/mL (ethyl acetate fraction), 0.64 ± 0.03 mg/mL (n-butanol fraction), and 2.27 ± 0.11 mg/mL (aqueous extracts) . ...
Article
Introduction: The genus Equisetum (Equisetaceae) is cosmopolitan in distribution, with 41 recognized species. Several species of Equisetum are widely used in treating genitourinary and related diseases, inflammatory and rheumatic problems, hypertension, and wound healing in traditional medicine practices worldwide. This review intends to present information on the traditional uses, phytochemical components, pharmacological activities, and toxicity of Equisetum spp. and to analyze the new insights for further study. Methods: Relevant literature has been scanned and collected via various electronic repositories, including PubMed, Science Direct, Google Scholar, Springer Connect, and Science Online, from 1960 to 2022. Results: Sixteen Equisetum spp. were documented as widely used in traditional medicine practices by different ethnic groups throughout the world. A total of 229 chemical compounds were identified from Equisetum spp. with the major group of constituents being flavonol glycosides and flavonoids. The crude extracts and phytochemicals of Equisetum spp. exhibited significant antioxidant, antimicrobial, anti-inflammatory, antiulcerogenic, antidiabetic, hepatoprotective, and diuretic properties. A wide range of studies have also demonstrated the safety of Equisetum spp. Conclusion: The reported pharmacological properties of Equisetum spp. support its use in traditional medicine, though there are gaps in understanding the traditional usage of these plants for clinical experiments. The documented information revealed that the genus is not only a great herbal remedy but also has several bioactives with the potential to be discovered as novel drugs. Detailed scientific investigation is still needed to fully understand the efficacy of this genus; hence, very few Equisetum spp. were studied in detail for phytochemical and pharmacological investigation. Moreover, its bioactives, structure-activity connection, in vivo activity, and associated mechanism of action ought to be explored further.
... Many authors in this context have reported the phytochemical composition of the poly-herbal compounds in relation to the wound healing potentials [7,15,17,22,25,29,35]. In this connection, many authors have reported secondary metabolites having an antioxidant nature, such as flavonoids and active phenol compounds present to be responsible for the wound healing process [8,18,24,29,71]. ...
... Many studies have suggested various targets for these active compounds that ultimately help to heal. These are mediated by multiple cascades, which includes mitogenic pathways [22,24,25,50], extracellular matrix synthesis pathway [11], free radical scavenging pathway [13,29e31,33,36,41], atherosclerosis pathway [19] and anti-inflammatory pathways [48,49,51]. It is therefore evident that the majorly studied pathway from the aforementioned is the free radical scavenging pathway. ...
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Background: The disruptions in skin integrity contribute to its disorientation, and tissue annihilations result in acute or chronic wound formation. Polyherbal formulations are widely used in traditional systems of mecdicine like ayurveda for wound healing. The combination of these traditional therapies with clinical therapies has helped in the development of various wound-healing products. Method: In this systematic review, the therapeutic potency of several polyherbal formulations from different medicinal floras is summed together in response to their impact on wound healing. The literature search was performed on Pubmed, Scopus, and ScienceDirect databases between 2010-2020. PRISMA methodology was applied to extract relevant information about polyherbal formulations. Result: A total of 54 articles were selected under all themes for the data extraction as per the PRISMA guidelines. These 54 articles have high-quality scores ≥3. Forty-three records were used for the narrative analysis, while nine records were used for the critical analysis in the narrative review. Further, theme-wise key data sets were screened from the selected literature and summarized in a tabular form. Bibliometric analysis of the Scopus database has also drawn attention to limited academic literature showcasing randomized clinical trials in the current subject. Most of these polyherbal formulations are tested in laboratory-scale studies, thus portraying further research options. Conclusion: Polyherbal formulations are effective in promoting the wound-healing process. They can stimulate a variety of physiological functions that accelerates the process of healing. These formulations merit further investigation in clinical trials, and production up scaling will aid in the creation of a new horizon of polyherbal wound healing products.
... Additionally, a few studies have demonstrated the antioxidant and wound healing efficacy of CA in vivo. The scratch assay using an HDF cell line is one of the most commonly used wound models for cell migration in cell culture studies [39,40]. ...
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Chlorogenic acid (CA) exhibits diverse biological activities, including antioxidant and antiinflammatory effects. This research aims to develop, optimize, and validate an HPLC method to quantify CA in methanol and investigate its in vitro proliferative and cell migration effects on human-dermal-fibroblast (HDF) cell lines in a dose-dependent manner. The HPLC experimental conditions were optimized using the central composite design (CCD) method for determining CA. Chromatographic separation occurred at a wavelength of 330 nm. Under the optimized conditions, the method exhibited linearity across a concentration range of 0.1-100 µg/mL, demonstrating sensitivity (LOQ:0.1µg/mL), precision (RSD%≤3.32), and accuracy (RE%≤4.05). To evaluate the in vitro proliferative and cell migration effects on HDFs, we employed the XTT cell proliferation assay and TAS-TOS commercial kits. The XTT assay revealed that CA displayed a proliferative effect within the concentration range of 75-250 µM (P <0.01), and at a concentration of 125 µM, TAS levels increased significantly (P<0.05). The scratch assay demonstrated that HDF cell migration increased at 12 h, with substantial closure of the wound area at 24 h when treated with CA concentrations between 75-125 µM. The results demonstrate that pure chlorogenic acid extracted from plants exhibits dose-dependent effects on cell proliferation, antioxidant, and cell migration
... Plant extracts containing many phenolic compounds have significant antioxidant, antibacterial and anti-inflammatory activity and are considered an effective option in the rapid repair of damaged skin tissues. In addition, it causes collagen synthesis by fibroblasts and helps increase the wound's strength [88]. Furthermore, optimal wound dressing should provide a moist environment to prevent dehydration and eliminate wound secretions [89]. ...
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Verbascum thapsus (V. thpsus), family Scrophulariaceae, has considerable importance in traditional medicine worldwide because of its antioxidant and anti-inflammatory activities. V. thpsus was used in traditional medicines as a useful drug for lung disease, sore throat, wound healing, and treatment of whooping cough. The aim of this study was to extract of V. thpsus bioactive fraction using antibacterial assay guided fractionation methodology and develop a system based on electrospun nanofibrous membrane (NFM) that can be effective by releasing the extract of V. thapsus for antibacterial and wound healing applications. For this purpose, the fractionation of total extract was done using Liquid-Liquid extraction method. The selected fraction based on its anti-bacterial activity was then subjected to the silica gel column chromatography for further purification. Since electrospinning is an economical and relatively simple method to produce continuous and uniform nanofibers, and due to its high specific surface area, adjustable pore size, and flexibility, special attention has been paid to loaded the most effective fraction on PVA nanofibers for applications such as wound dressings. The obtained result showed that, the purified V. Thapsus extract has a concentration-dependent antimicrobial activity against Escherichia coli and Staphylococcus aureus. The phytochemically analyses of bioactive fraction by High- performance liquid chromatography (HPLC) proved the presence of 6 phenolic acids, 4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, and flavonoid, rutin, as the major compounds. Also, physicochemical characterization of PVA-selected extract loaded electrospun nanofibrous membranes (NFM) were analyzed by scanning electron microscope (SEM), Fourier transform infrared spectrometer (FT-IR). MTT and hemolysis assays were done to affirm the biocompatibility of fabricated scaffolds. Release profile of extract loaded- NFM showed continues release of extract from mat during 90h. Moreover, the capability of these NFM in wound healing application was evaluated in-vitro and in-vivo. The cell viability test (MTT), cell adhesion images, antioxidant, antibacterial, hemolysis assays and in-vitro and in-vivo wound healing assays confirmed that fabricated NFM containing 5 % butanolic extract were the most
... In addition, oxidative stress is considered a precursor to senescence and cell death, prolonging inflammation and cell migration. Nonetheless, the presence of exogenous antioxidant agents, such as some secondary metabolites of plants, contributes to modulating the production and destruction of ROS [19,23,24]. ...
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Diabetes mellitus (DM) affects the wound healing process, resulting in impaired healing or aberrant scarring. DM increases reactive oxygen species (ROS) production, fibroblast senescence and angiogenesis abnormalities, causing exacerbated inflammation accompanied by low levels of TGF—β and an increase in Matrix metalloproteinases (MMPs). . Propolis has been proposed as a healing alternative for diabetic patients because it has antimicrobial, anti-inflammatory, antioxidant and proliferative effects and important properties in the healing process. An ethanolic extract of Chihuahua propolis (ChEEP) was obtained and fractionated, and the fractions were subjected to High–Performance Liquid Chromatography with diode–array (HPLC–DAD), High–Performance Liquid Chromatography–Mass Spectrometry (HPLC–MS) and Gas Chromatography‒Mass Spectrometry (GC–MS) analyses and 46 compounds were detected. Deep wounds were made in a murine DM model induced by streptozotocin, and the speed of closure and the wound tensile strength were evaluated by the tensiometric method, which showed that ChEEP had similar activity to Recoveron, improving the speed of healing and increasing the wound tensile strength needed to open the wound again. A histological analysis of the wounds was performed using H&E staining, and when Matrix metalloproteinase 9 (MMP9) and α—actin were quantified by immunohistochemistry, ChEEP was shown to be associated with improved histological healing, as indicated by the reduced MMP9 and α—actin expression. In conclusion, topical ChEEP application enhances wound healing in diabetic mice.
... Chlorogenic acid has been shown to enhance capillary density and promote collagen production. Along with its free radical scavenging properties, it exerts anti-oxidant and anti-inflammatory on the metalloproteinases in the ECM of the injured tissues during second-degree burns [35,36]. ...
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Background and Aim Potato (Solanum tuberosum L.) is mainly characterized by its antioxidant and healing properties. Therefore, this study aimed to evaluate the effects of an ointment based on S. tuberosum L. “papa tumbay” on burns induced in Balb/c mice (Mus musculus). Materials and Methods The experimental animals were divided into four groups (n = 5/group) 48 h before second-degree burns were inducted. After epilating the loin areas of the mice and anesthetizing them with ketamine/xylazine (80 mg/kg/10 mg/kg) through intraperitoneal (i.p.) route, a round metal rod (0.7 cm in diameter) was placed on the depilated skin at a temperature of 100°C for 5 s. Group I was not given any treatment, Group II was treated with silver sulfadiazine (1%), and the other two groups (III and IV) were treated with the ointment formulated based on S. tuberosum L. “papa tumbay” at 1% and 2%, respectively. After performing the treatment for 21 days, the mice were euthanized using i.p. sodium pentobarbital (185 mg/kg) to obtain skin samples. The samples were preserved in 10% neutral-buffered formalin and subjected to histopathological analysis. Results We found statistically significant differences in the histopathological sections between the groups (p < 0.05). The abundant collagen and fibroblasts observed in the direction of the dermis in Groups III and IV indicate that the phytoconstituents present in the potato might promote the healing of the second-degree burns until day 21 of treatment. Conclusion Our findings showed that the ointments based on the ethanolic extracts of S. tuberosum L. “papa tumbay,” especially the 2% ointment, might accelerate the healing of second-degree burns induced in Balb/c mice.
... However, if the endogenous antioxidant system fails to balance the production and degradation of ROS, the excessive levels of ROS create a condition of oxidative stress, which activates pro-apoptotic proteins, generating toxic effects on cells, causing inflammation and delay in the healing process. Exogenous antioxidants such as those found in plant metabolites are an alternative to reduce oxidative damage, having a beneficial effect on wound healing [32,[68][69][70][71][72]. In previous studies carried out in our laboratory, the antioxidant capacity (SA 50 ) of ChEEP was determined, and the extract showed an SA 50 = 15.75 µg/mL [27], in agreement with the antioxidant activity index is considered a very strong antioxidant activity [73]. ...
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Skin wound healing is a complex biochemical process of tissue repair and remodeling in response to injury. Currently, the drugs used to improve the healing process are inaccessible to the population, are costly, and have side effects, making the search for new treatment alternatives necessary. Propolis is a natural product produced by bees that is widely recognized and used in folk medicine for its multiple biomedical activities. However, therapeutic information regarding Mexican propolis is limited. This study aimed to evaluate the wound-healing effect of the Chihuahua ethanolic extract of propolis (ChEEP). Macroscopic and histological analyses were performed using a mouse wound-healing model. The topic acute toxicity assay showed that propolis at 10% w/v had no toxic effects. ChEEP has antibacterial activity against the Gram-positive bacteria Staphylococcus aureus and Staphylococcus epidermidis. Moreover, it exhibited good anti-inflammatory activity evaluated through mouse ear edema induced by 12-O-tetradeca-noylphorbol-13-acetate (TPA). A full-thickness incision lesion was created in mice and treated topically with 10% ChEEP. At Day 14 post-treatment, it was observed that propolis increased wound contraction and reduced healing time and wound length; furthermore, propolis increased the tensile strength of the wound, as determined with the tensiometric method, and promoted the formation of type I collagen at the site of injury, as evaluated with Herovici stain. These findings suggest that the topical administration of ChEEP can improve skin wound healing, probably due to the synergistic effect of its components, mainly polyphenols, in different steps of the wound-healing process. It should be noted this is the first time that the wound-healing activity of a Mexican propolis has been evaluated.
... In clinical trials, topical phytotherapy combining E. arvense extracts with other herbal extracts showed benefits to brittle nail disorder. Ointment containing E. arvense (3%) showed wound-healing effects [28]. Other clinical trials reported the efficacies of E. arvense for benign prostate hyperplasia and chronic musculoskeletal pain [29]. ...
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Trends in skin and hair treatments focus on natural products due to undesired effects of chemically synthetic ingredients. This study aims to investigate the cosmeceutical effects of Equisetum debile (horsetail) extracts relating to anti-hyperpigmentation via tyrosinase, anti-wrinkle formation via matrix metalloproteinases (MMPs), and anti-androgenic alopecia via 5α-reductase. Ethanolic extracts were sequentially partitioned into semi-purified fractions hexane, dichloromethane, ethyl acetate, methanol, and methanol insoluble residue. The ethyl acetate fraction possessed the highest total phenolic content (39.24 ± 0.72 mg gallic acid/g), the strongest anti-tyrosinase activities (583.33 ± 23.59 mg kojic acid/g), and potent collagenase inhibitions (IC50 MMP-1 and MMP-2 of 0.82 ± 0.09 and 0.94 ± 0.11 mg/mL, respectively). All extracts showed considerable inhibitions of 5α-reductase ranging from 44.59 ± 0.40 to 83.07 ± 3.46% with the strongest activity from the dichloromethane fraction (1.48 ± 0.06 mg finasteride/g). In conclusion, E. debile extracts exhibit cosmeceutical potentials. This study suggests that the E. debile ethyl acetate fraction could be used as a promising ingredient to organically treat hyperpigmentation and delay the skin aging process. In addition, compared to the current recommended intake of finasteride (1 mg/day) for androgenic alopecia, the dichloromethane fraction is proposed as an alternative source to naturally remediate hair loss.
... Different botanical drug formulations of C. arvensis, L. stoechas, and H. italicum exhibited in vitro regenerative properties as these increase the fibroblast proliferation and migration [93]. A botanical drug consisting of Achillea millefolium L., Hyssopus officinalis L., Equisetum arvense L. and Echinacea purpurea L. showed increased collagen synthesis activity by mouse fibroblasts in vitro, thereby suggestive of wound healing capacity [94]. Another botanical drug consisting of Vitex negundo L., Emblica officinalis Gaertn, and Tridax procumbens L. had demonstrated wound healing activity by rapid movements of keratinocytes and fibroblasts to the wound site, increased rates of skin regenerations, wound contraction, and collagen synthesis [95]. ...
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Skin, the largest organ of the body, plays a vital role in protecting inner organs. Skin stem cells (SSCs) comprise a group of cells responsible for multiplication and replacement of damaged and non-functional skin cells; thereby help maintain homeostasis of skin functions. SSCs and differentiated cells of the skin such as melanocytes and keratinocytes, have a plethora of applications in regenerative medicine. However, as SSCs reside in small populations in specific niches in the skin, use of external stimulants for cell proliferation in vitro and in vivo is vital. Synthetic and recombinant stimulants though available, pose many challenges due to their exorbitant prices, toxicity issues and side effects. Alternatively, time tested traditional medicine preparations such as polyherbal formulations are widely tested as effective natural stimulants, to mainly stimulate proliferation, and melanogenesis/prevention of melanogenesis of both SSCs and cells of skin origin. Complex, multiple targets, synergistic bioactivities of the phytochemical constituents of herbal preparations amply justify these as natural stimulants. The use of these formulations in clinical applications such as in skin regeneration for burn wounds, wound healing acceleration, enhancement or decrease of melanin pigmentations will be in great demand. Although much multidisciplinary research is being conducted on the use of herbal formulas as stem cell stimulants, very few related clinical trials are yet registered with the NIH clinical trial registry. Therefore, identification/ discovery, in depth investigations culminating in clinical trials, as well as standardization and commercialization of such natural stimulants must be promoted, ensuring the sustainable use of medicinal plants. Graphical Abstract
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Este estudo foi conduzido para verificar o efeito do extrato aquoso e da pomada à base de casca de Caryocar brasiliense sobre a retração de feridas em coelhos. Foram utilizados 12 coelhos Nova Zelândia Branco divididos em dois grupos e quatro lesões foram produzidas na região dorsal de cada animal. As lesões à direita foram tratadas com extrato aquoso (grupo 1) ou com a pomada (grupo 2) e as lesões à esquerda foram tratadas com solução salina 0,9% (grupo controle). Os níveis de colágeno e fibroblastos foram menores (P<0,05) em lesões tratadas com extrato aquoso, comparado com o grupo controle. Aos 7 e 14 dias após o procedimento, a retração das lesões era maior (P<0,05) quando tratadas com extrato aquoso e nas lesões tratadas com a pomada, a melhora ocorreu apenas no 7º dia, comparado com o tratamento controle. Concluiu-se que o extrato aquoso de casca de Caryocar brasiliense melhorou a retração de feridas por um período de tempo maior do que a pomada.
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p>Data generated through systematic investigation, carried out on the evaluation of phyto-extracts on wound healing research during the last 20 years have been compiled. About 450 plant species having wound healing properties have been identified. The present knowledge of the wound healing process comprise coagulation, inflammation, proliferation, formation and accumulation of fibrous tissues, collagen deposition, epithelialization, contraction of wound with formation of granulation tissues, remodeling and maturation. The constituents of the plant extracts modulate one or more of the above stages. It was the endeavor to identify the active constituents responsible for antimicrobial activity, free radical scavenging properties, stimulators of enhanced collagen production and/or angiogenesis promoters with identification of lead scaffold chemical structures. Multiple phytochemicals concentrated and blended in optimal concentrations, are expected to be available in future years to carry out multi-tasking efforts in wound healing as more knowledge about the properties of the key constituents are unveiled. This article is open to POST-PUBLICATION REVIEW . Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.</p
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A wound is a disruption of the normal anatomical structure and function of a tissue. Wounds cure in an orderly and timely repair process which is characterized by thr-ee dynamic and interactive phases: inflammation, proliferation and the remodeling. The present review was designed to elaborate the cellular and molecular targets for plant secondary metabolites that target the various aspects of wound repair process. The common mechanism of action of natural products established through in vitro and animal studies include direct action on skin cells regeneration, increase in con-nective tissue deposition, antioxidant activity, inflammatory cells activity and mod-ulation of cytokine and growth factor production and/or function. All these demon-strated pharmacological effects could be exploited to overcome an acute or path-ological wound healing conditions. The therapeutic potential of various chemical classes of natural products that act through one or multiple targets are discussed.
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Publisher Summary This chapter discusses the analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Analyses of the Folin-Ciocalteu (FC) type are convenient, simple, and require only common equipment and have produced a large body of comparable data. Under proper conditions, the assay is inclusive of monophenols and gives predictable reactions with the types of phenols found in nature. Because different phenols react to different degrees, expression of the results as a single number—such as milligrams per liter gallic acid equivalence—is necessarily arbitrary. Because the reaction is independent, quantitative, and predictable, analysis of a mixture of phenols can be recalculated in terms of any other standard. The assay measures all compounds readily oxidizable under the reaction conditions and its very inclusiveness allows certain substances to also react that are either not phenols or seldom thought of as phenols (e.g., proteins). Judicious use of the assay—with consideration of potential interferences in particular samples and prior study if necessary—can lead to very informative results. Aggregate analysis of this type is an important supplement to and often more informative than reems of data difficult to summarize from various techniques, such as high-performance liquid chromatography (HPLC) that separate a large number of individual compounds .The predictable reaction of components in a mixture makes it possible to determine a single reactant by other means and to calculate its contribution to the total FC phenol content. Relative insensitivity of the FC analysis to many adsorbents and precipitants makes differential assay—before and after several different treatments—informative.
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Chlorogenic acids are polyphenolic compounds that occur ubiquitously in foods of plant origin. They are quinic acid esters of hydroxycinnamic acid.Recently, naturally occurring plant phenolics have attracted considerable attention in relation to their physiological potential. Depending upon the conditions, phenolic compounds can be either beneficial or detrimental to biological processes.We comprehensively summarized chlorogenic acids and related compounds in absorption, metabolism and biological activity. Chlorogenic, caffeic and quinic acids are well absorbed in humans and rats. Metabolic transformations of chlorogenic acids in the human system may be crucial for their biological effect.The antioxidant activities of chlorogenic acids are preserved by inhibiting the formation of reactive oxygen species or by scavenging them. As a result, chlorogenic acidsmay play beneficial role in the prevention of certain oxidative diseases. The observed pharmacological activities of medicinal plants relate to the chlorogenic acidsconstituents of them. Despite the abundance of biological data demonstrating the antioxidant activities of chlorogenic acids, it remains controversial whether these compounds are potent antioxidants or pro-oxidants. Chlorogenic and caffeic acids switch from anti- to pro-oxidant activity, depending on their concentration, on the presence of free transition metal ions, or on their redox status.