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Wound Healing Activity of Extracts of Malva sylvestris and Stachys lavandulifolia

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The flowers of Stachys lavandulifolia Vahl (Lamiaceae) and Malva sylvestris Linn (Malvaceae) are traditionally used to treat various skin disorders, antimicrobial and anti-inflammatory. The chloroform extract of M. sylvestris flowers and aqueous extract of S. lavandulifolia flowers were used to evaluate the wound healing activity at 200 mg/kg/day dose. Wounds were induced in Wistar rats divided into four groups as following; Group-I was treated with cold cream. Groups-II and -III were treated with cold cream containing of extracts, Group-IV received the standard drug (nitrofurazone). The efficacy of treatment was evaluated based on wound area and histopathological characteristics. The extract-treated animals by M. sylvestris showed significant reduction in the wound area when compared with other groups. Also, histopathological studies of the tissue obtained on days 6 th , 9 th and 16 th from the extract-treated by M. sylvestris showed increased well organized bands of collagen, more fibroblasts and few inflammatory cells.
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www.ccsenet.org/ijb International Journal of Biology Vol. 3, No. 1; January 2011
ISSN 1916-9671 E-ISSN 1916-968X
174
Wound Healing Activity of Extracts of Malva sylvestris and Stachys
lavandulifolia
Abdollah Ghasemi Pirbalouti (Corresponding author)
Researches Centre of Medicinal Plants & Ethno-veterinary
Islamic Azad University-Shahrekord Branch, Shahrekord, POBox: 166, Iran
Tel: 98-381-336-1060 E-mail: ghasemi@iaushk.ac.ir
Abed Koohpyeh
Researches Centre of Medicinal Plants & Ethno-veterinary
Islamic Azad University-Shahrekord Branch, Shahrekord, POBox: 166, Iran
The research is financed by Islamic Azad University-Shahrekord Branch (Sponsoring information)
Abstract
The flowers of Stachys lavandulifolia Vahl (Lamiaceae) and Malva sylvestris Linn (Malvaceae) are traditionally
used to treat various skin disorders, antimicrobial and anti-inflammatory. The chloroform extract of M. sylvestris
flowers and aqueous extract of S. lavandulifolia flowers were used to evaluate the wound healing activity at 200
mg/kg/day dose. Wounds were induced in Wistar rats divided into four groups as following; Group-I was treated
with cold cream. Groups-II and -III were treated with cold cream containing of extracts, Group-IV received the
standard drug (nitrofurazone). The efficacy of treatment was evaluated based on wound area and
histopathological characteristics. The extract-treated animals by M. sylvestris showed significant reduction in the
wound area when compared with other groups. Also, histopathological studies of the tissue obtained on days 6th,
9th and 16th from the extract-treated by M. sylvestris showed increased well organized bands of collagen, more
fibroblasts and few inflammatory cells.
Keywords: Wound healing, Medicinal plants, Stachys lavandulifolia, Malva sylvestris
1. Introduction
Approximately one-third of all traditional medicine in use are for the treatment of wounds and skin disorders,
compared to only 1-3% of modern drugs (Mantle et al., 2001). Reports about medicinal plants affecting various
phases of the wound healing process, such as coagulation, inflammation, fibroplasia, epithelization,
collagenation and wound contraction are abundant in the scientific literature (Asif et al., 2007; Hemmati &
Mohammadian, 2000; Khalil et al., 2006; Nayak et al., 2007). A survey of the ethnobotanical studies, carried out
in Iran, indicated the use of several of plant species by the inhabitants of the area, especially by those habiting
the rural areas for wound healing purpose (Ghasemi Pirbalouti, 2009a; Ghorbani, 2005; Zargari, 1990).
Malva sylvestris Linn (Malvaceae), known locally as “Panirak”, is a medicinal plants in Iran whose flowers are
used for the treatment of various ailments, including cold, cough and burn and cut wound healing in rural areas
of Iran (Ghasemi Pirbalouti, 2009a; Ghorbani, 2005; Zargari, 1990). Fluidextract of M. silvestris flowers and
leaves are used as a valuable remedy for cough and inflammatory diseases of mucous membranes (Farina, 1995).
A new anthocyanin, malvidin 3-(6-malonylglucoside)-5-glucoside has been characterized in both wild and
cultivated forms of M. Sylvestris (D’Amelio, 1999; Takeda et al., 1989). The malvone A (2-methyl-3-methoxy-5,
6-dihydroxy-1, 4-naphthoquinone) is reported (Cutillo et al., 2006; Veshkurova et al., 2006)
Stachys lavandulifolia Vahl (Lamiaceae) a well-known traditional herb used in tribal medicine of Iran is locally
known as “Chaye-e-Kohi or Lolopashmak”. The decoction of the flowers is being used by the tribal people for
treatment of skin infection, menorrhagia and anti-bacterial (Ghasemi Pirbalouti, 2009b; Zargari, 1990).
No systematic studies have yet been carried out on the clinical evaluation of the wound healing potency of M.
sylvestris and S. lavandulifolia so its effects were investigated using wound are and histopathological
characteristics in rats.
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Published by Canadian Center of Science and Education 175
2. Materials and Methods
2.1 Plant material and extract preparation
The flowers of M. sylvestris and S. lavandulifolia were collected from mountain areas of Zagross, district of
Chaharmahal va Bakhtiari, Iran, during May–June, 2008. Their identity was confirmed and voucher specimens
were deposited at the spice, aromatic and medicinal plant research centre (SAMPRC), Islamic Azad University,
Iran (Mozaffarian, 1996). Powdered flowers of M. sylvestris (300 g) were Soxhlet-extracted with 80%
chloroform (Merck, Germany) for 6 h (yield: 6%). The flower powder (200 g) of S. lavandulifolia was refluxed
with 750mL of double distilled water for 2 h at 70 °C. The infusions were filtered and concentrated under rotary
vacuum (model Zirbus 302®) for about 1 h (yield: 5.5%).
2.2 Animals
Male Wistar rats (180-200 g) of 2-3 months were used. The animals were housed in standard environmental
conditions of temperature (22 ±3ºC), humidity (60 ±5%) and a 12 h light/dark cycle. During experimental time
rats were given standard pellet diet (Pastor Institute, Iran) and water ad libitum.
2.3 Wound healing activity
Wound induction and evaluation extracts for properties wound healing before the beginning of the wound
healing experiments, the dorsal skin of the Wistar rats were shaved. Animals were anesthetized with 1.5mg/kg
i.p. of Ketamin and Xylazine. A full thickness of the excision wound (circular area about 150 mm2 and 2 mm
depth) was created along the markings using toothed forceps, a surgical blade and pointed scissors (Khalil et al.,
2006).
The animals were divided randomly into three groups of nine each. Group-I was treated with cold cream
(Control). Groups-II and -III were treated topically with cold cream prepared from aqueous extract of S.
lavandulifolia and M. sylvestris (200 mg/kg/day) respectively, Group-IV received the standard drug
(nitrofurazone).
During the wound healing period and at the present time intervals, the wound area was traced manually and
photographed. The wound area was calculated using AutoCAD Version 14 (Autodesk Company) software. At days 6th,
9th and 16th the experiment was terminated and the wound area was removed from the surviving animals for
histological examination. The excision skin biopsies were fixed in 4 % formaldehyde solution 48 h during the
experimentation period.
2.4 Analysis of data
The relative burn wound area was statistically analyzed as mean ±S.D and statistically significance between
treated and control groups were analyzed by means of Student’s t-test. Data are significant;
P-values d0.05 compared with control by the program “SAS ver 6.12 full”.
3. Results
The animals treated with the M. sylvestris showed a significant reduction in the wound area when compared with
other groups (Table 1). The animals treated with the extract of M. sylvestris showed faster epithelialization than
those treated with the standard drug and aqueous extract of
S. lavandulifolia. The extract-treated animals by M. sylvestris showed about 99% reduction in the wound area
when compared with nitrofurazone and S. lavandulifolia extract which were 95 and 92%, respectively (Table 1).
The study of the histological structure showed the tissue regeneration was grater in the skin wound treated with
cold cream containing the M. sylvestris extract and following nitrofurazone ointment and
S. lavandulifolia extract (Table 2 and Fig 1). The skin wound treated with cold cream (control) presented edema,
monocyte cells and area with cellular necrosis that were not observed in the treated with herbal ointments and
standard drug (Table 2 and Fig 1).
4. Discussions and Conclusions
Wound healing is a process by which damaged tissue is restored as closely as possible to its normal state and
wound contraction is the process of shrinkage of the area of the wound (Nayak et al., 2007). It is mainly
dependent upon the type and extent of damage, the general state of health and the ability of the tissue to repair.
Despite the traditional uses M. sylvestris and S. lavandulifolia in wound healing process in Iran, there are no
reported data available in the literature. M. sylvestris and S. lavandulifolia widely distributed plants of Iran are
used for the infectious, anti-inflammatory, anti-microbial, skin disease and for wound healing properties
according to several ethnobotanical surveys (Ghasemi Pirbalouti, 2009a,b; Ghorbani, 2005; Zargari, 1990).
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In present study, results of wound area measurements, as shown in table 1, indicated a healing potential for the
M. sylvestris extract. Statistically, area measurements showed that there is significance between the different
groups.
Wound healing is a very complex, multifactor sequence of events involving several cellular and biochemical
processes (Philips et al., 1991). The aims in these processes are to regenerate and reconstruct the disrupted
anatomical continuity and functional status of the skin (Philips et al., 1991). The results in this study are in
support that wound healing and repair is accelerated by applying M. sylvestris which was high-lighted by the full
thickness coverage of the wound area by an organized epidermis in the presence of mature scar tissue in the
dermis. This ability was especially obvious when the data were compared with the other groups. The results of
histological evaluation showed that M. sylvestris significantly increased the rate of wound contraction and
collagen turnover. Collagen, the major component which strengthens and supports extracellular tissue, is
composed of the amino acid, hydroxyproline, which has been used as a biochemical marker for tissue collagen
(Philips et al., 1991).
The preliminary phytochemical analysis of the flower extract by researchers showed the absence of anthocyanin,
malvin, malvidin 3-(6-malonylglucoside)-5-glucoside, malvaline, niacin and folic acid. Any one of the
phytochemical constituents (malvone A: 2-methyl-3-methoxy-5, 6-dihydroxy-1,4-naphthoquinone) present in M.
sylvestris may be responsible for antimicrobial activity (Cutillo et al., 2006; D’Amelio, 1999). It may be either
due to the individual or additive effect of the phyto-constituents that hastens the process of wound healing. The
exact component of the extract that is responsible for this effect, however, was not investigated. Further
phytochemical studies are needed to isolate the active compound(s) responsible for these pharmacological
activities.
In conclusion, while plant based traditional medicine has been used throughout generations, the efficiency of
such treatments requires experimental backup and scientific verification. In this study, two plant species
presented were selected based on ethnopharmacological information, provided by local communities. This study
confirms the wound healing activity of the flowers of M. sylvestris. Further studies need to be done to identify
and separate the group of active constituents responsible for anti-inflammatory activity and wound healing
activity from alcohol and petrol ether extracts.
Acknowledgements
This work was supported by IAUSHK (Researches Affairs). The authors are thankful to Mr. F. Fadi Fard, Ph.D
and Mr. M. Farid, Ph.D (Surgical) for their technical support.
References
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Table 1. Effect of the treatments on wound healing in rats
Wound area relative (cm2)
Treatments
16th 9th 6th
0.117±0.4 * 0.39±0.21 * 0.78±0.22 ** S. lavandulifolia + Cold cream
0.23±0.09 ** 0.84±0.05 * 0.87±0.05 ** M. sylvestris + Cold cream
0.077±0.04 ** 0.33±0.13 ** 0.77±0.15 ** Nitrofurazone
0.23 1.132 1.38
Control (Cold cream)
Each value represents mean ±S.D. N= 9 animals.
**: P
d
0.01, *: P
d
0.05 levels of significance.
Table 2. Effect of the treatments on the evolution of wounds in rats after 6, 9 and 16 days of topical application
Treatments
Inflammatory
cells
Collagen
fibers
Re-epithelization
Organization of the
collagen
Necrosis Fibrin
6 9 16 6 9 16 6 9 16 6 9 16 6 9 16 6 9 16
Control
(Cold cream )
+++ ++ ++ _ _ + _ _ + _ _ + +++ ++ + +++ ++ +
Standard drug
(Nitrofurazone)
+ + _ + + ++ + + ++ + ++ ++ + _ _ ++ + _
S. lavandulifolia + + + + + ++ + + + + + ++ + + _ ++ + _
M. sylvestris + _ _ + ++ +++ + + +++ ++ ++ +++ + _ _ + + _
+: slight, ++: moderate, +++: extensive, -: absent.
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Figure 1. Flowers and areal plant of Malva sylvestris and Stachys lavandulifolia
Nitrofurazone (a)
M. sylvestris + cold cream (b)
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Published by Canadian Center of Science and Education 179
Stachys lavandulifolia (c)
Figure 2. Histological evaluation after 16 days of wound creation in three groups (a, b & c)
... The use of Malva sylvestris, a species of the mallow genus Malva in the family, has been documented since long ago. M. sylvestris is recommended for acne and skincare, as an antiseptic and emollient [192][193][194], and as an antimicrobial and anti-inflammatory agent for burn and cut wound healing [195][196][197]. The healing capabilities of this plant relate to the mucilage and flavonoids found in the leaves and flowers [198]. ...
... The healing capabilities of this plant relate to the mucilage and flavonoids found in the leaves and flowers [198]. Indeed, M. sylvestris flowers extract contains anthocyanin, malvidin, flavones, flavonols, malvin, malvaline, niacin, and folic acid, which are responsible for their pharmacological and biological activities [197][198][199] (Figure 3). ...
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A new anthocyanin, malvidin 3-(6″-malonylglucoside)-5-glucoside has been characterized in both wild and cultivated forms of Malva sylvestris. Thus the classic source of the anthocyanin, malvin, actually contains the pigment in the flowers in malonated form. Malonated anthocyanins were also detected in Althaea rosea, Lavatera olbia and a Sphaeralcea sp. but they were not present in five other species in the family.
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The object of this study was to examine the initiation and pattern of capillary growth associated with wound healing. Collagen sponges were implanted subcutaneously in the hind limbs of adult male rats to stimulate the formation of granulation tissue. Blood vessels of the hind limbs of euthanized rats were perfused with Mercox (an acrylic monomer) via the abdominal aorta at selected periods of time following sponge implantation. When the perfusate was completely cured, the sponge and parajacent tissues were excised and subsequently macerated by alternating immersion in 40% KOH and distilled water. Cast replicas of the vascular lumina were coated with gold and imaged by scanning electron microscopy. At 6 hr, punctate depressions at the periphery of the replicas of vein and venule lumina were noted. The depressions represented sites of leukocyte margination. By 24 hr, the depressions increased numerically, indicating a great increase in the sites of leukocyte margination. The number of these depressions decreased by 48 hr. Concomitantly, the depressions representing endothelial cell nuclei became more pronounced, indicating nuclear hypertrophy of these cells. In addition, capillary bud formation was initiated. At 72 hr, capillary buds were quite apparent and arose solely from venules. Between 7 and 14 days, replicas of capillary lumina were longer and formed an elaborate network, presumably by end-to-end, side-to-side, and end-to-side anastomoses. The network was formed circumferential to the sponge and then capillary sprouts entered the sponge's interstitial spaces.
Article
Aqueous alcoholic mallow flower extracts were analyzed both by HPTLC-densitometry in the reflectance mode at 530 nm and by reversed-phase HPLC with gradient elution. For the mallow flower anthocyanins the best chromatographic resolution was obtained by HPLC, which revealed only two main compounds, confirmed by FAB-MS: malvidin 3,5-O-diglucoside (malvin) and malvidin 3-O-(6"-O-malonylglucoside)-5-O-glucoside. The HPTLC densitometric method on cellulose plates provides accuracy, reproducibility and selectivity for the quantitative analysis of the anthocyanins and this method was shown to be much more sensitive than the HPLC-DAD system, at 530 nm. Both methods give comparable quantitative results for total anthocyanins when applied to mallow flowers from two different sources: Italy and Albania.
Article
Plants are of relevance to dermatology for both their adverse and beneficial effects on skin and skin disorders respectively. Virtually all cultures worldwide have relied historically, or continue to rely on medicinal plants for primary health care. Approximately one-third of all traditional medicines are for treatment of wounds or skin disorders, compared to only 1-3% of modern drugs. The use of such medicinal plant extracts for the treatment of skin disorders arguably has been based largely on historical/anecdotal evidence, since there has been relatively little data available in the scientific literature, particularly with regard to the efficacy of plant extracts in controlled clinical trials. In this article therefore, adverse and beneficial aspects of medicinal plants relating to skin and skin disorders have been reviewed, based on recently available information from the peer-reviewed scientific literature. Beneficial aspects of medicinal plants on skin include: healing of wounds and burn injuries (especially Aloe vera); antifungal, antiviral, antibacterial and acaricidal activity against skin infections such as acne, herpes and scabies (especially tea tree (Melaleuca alternifolia) oil); activity against inflammatory/immune disorders affecting skin (e.g. psoriasis); and anti-tumour promoting activity against skin cancer (identified using chemically-induced two-stage carcinogenesis in mice). Adverse effects of plants on skin reviewed include: irritant contact dermatitis caused mechanically (spines, irritant hairs) or by irritant chemicals in plant sap (especially members of the Ranunculaceae, Euphorbiaceae and Compositae plant families); phytophotodermatitis resulting from skin contamination by plants containing furocoumarins, and subsequent exposure to UV light (notably members of the Umbelliferae and Rutaceae plant families); and immediate (type I) or delayed hypersensitivity contact reactions mediated by the immune system in individuals sensitized to plants or plant products (e.g. peanut allergy, poison ivy (Toxicodendron) poisoning).
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This paper is the result of ethnobotanical survey on the Turkmens of Golestan and Khorasan Province (Iran) conducted from June 2002 to the end of 2003. Turkmens are traditionally an isolated ethnic group residing in northern parts of Iran. We studied the folk herbal medicine among Turkmens of Iran. Totally, 136 species from 51 families were documented from which 120 species used as medicinal and 84 species mentioned by three or more informants. Information about plant uses is all summarized in Table 1. Some interesting and endemic species have been reported for medicinal uses, also some new uses for common species were documented. Some of these species are good targets for further analysis.
Article
A sesquiterpene and a tetrahydroxylated acyclic diterpene as well as two known monoterpenes, 6 C(13)nor-terpenes and 11 aromatic compounds were isolated from the water extract of Malva silvestris. The structures of the compounds were determined by spectroscopic NMR and MS analysis. Effects of these compounds on germination and growth of dicotyledon Lactuca sativa L. (lettuce) were studied in the 10(-4)-10(-7)M concentration range.
Article
The wound healing effect of the aqueous extracts of Inula viscosa, Ajuga chia, Rubia taenifolia and Parieteria diffusa, and the oil of Laurus nobilis, dispersed in water, were examined. The 10% (w/w) Pluronic F127 (PF127) was added to the applied preparations, in order to modify the aqueous extracts viscosity, and to stabilize the oil dispersion. A full thickness wound was made in the dorsal area of the mice. The wounds were treated with the different preparations with 12h intervals for four times in two successive days. For 16 days, the wounds were visually observed, photographically documented and the wound area was measured. After day 16, the animals were sacrificed and the histology of the wound area was examined. The best wound healing activity was observed with the extract of Inula viscosa, followed by Parieteria diffusa, Laurus nobilis, Ajuga chia and the least active extract was that of Rubia taenifolia.
Article
The isolation and structure of a phytoalexin, malvone A (2-methyl-3-methoxy-5,6-dihydroxy-1,4-naphthoquinone) is reported. Malvone A formation is induced in Malva sylvestris L. by the plant pathogen Verticillium dahliae. In a turbimetric assay for toxicity to V. dahliae, it had an ED50 value of 24 microg/ml. The structure of malvone A was determined by MS and NMR spectroscopy, and by X-ray crystallographic analysis. The X-ray analysis showed water molecules were located in channels that run along the a-axis.