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B. Shivananda, S. Sivachandra Raju, A.V. Chalapathi Rao
Journal of Wound Care, Vol. 16, Iss. 7, 01 Jul 2007, pp 298 - 302

The therapeutic efficacy of indigenous plants has been described by traditional herbalists1 The past decade has seen a considerable change in opinion on ethno­pharmacological therapeutic applications, and the presence of various life-sustaining constituents in plants has prompted researchers to examine them with a view to determining their potential wound-healing activities.1
Matricaria recutita L. (M. recutita), also known as German chamomile, Matricaria chamomilla, Chamomilla chamomilla, Matricaria suaveolens and chamomile (also spelt as camomile), has been used for centuries as a medicinal plant, mostly for gastrointestinal ailments and skin injuries and problems.2 In Germany it is used in wound care, where its effectiveness has been reported.3 Aromatherapy with chamomile essential oil has been found to be effective in healing episiotomy wounds.4
The controlling effect of chamomile flower extract on menopausal symptoms5 indicates its oestrogenic activity. Oestrogen receptors have been detected in the skin.6 Oestrogen is a proinflammatory7 and so promotes wound healing.8,9
The beneficial effects of chamomile flower extract on aphthous stomatitis10 and methotrexate-induced oral ulcers11 could be due to its immunostimulating or immunomodulating properties. Its antipruritic effect12 may be due either to its antagonistic action on histamine or serotonin receptors, or to an anti-ulcerogenic effect13 arising from its antagonistic action on H2 receptors. Histamine-receptor antag­onists accelerate skin-barrier repair14 and the 5HT2 receptor antagonist, ketanserin, promotes wound healing.15
Chamomile is also reported to have opioid-like16 properties. When applied topically, opioids accelerate wound healing17 and potentiate platelet aggregation.18 Platelet reaction and activation initiate the wound-healing process.
However, there are no scientifically proven data to support the wound-healing activities of M. recutita. This study therefore aimed to explore the effects of M. recutita flower extract on wound healing.
Method
Plant material and extract preparation
Blended flower powder of M. recutita (Liberty Richter, New Jersey, USA) was procured from a herbal pharmacy in Trinidad. The fine powder (60g) was suspended in 200ml of deionised water and kept in a water bath at 40°C for 24 hours. The mixture was filtered first with a fine muslin cloth and then with filter paper (Whatman no. 1). The clear filtrate was dried in a water bath at 40°C, and the clear paste obtained (10g) was used for the study. The extract was subjected to preliminary phytochemical tests.
Animals
The study was approved by the ethics committee for animal experimentation (AHC06/07/1) at the University of the West Indies, St Augustine, Trinidad.
An acute toxicity study was conducted to determine the dose. Rats of either sex were fed with increasing doses (1, 2, 4, and 8g/kg of body weight) of the extract, dissolved in water, for 14 days. Doses of up to 4g/kg body weight did not produce any signs of toxicity and mortality. The animals were physically active and consuming food and water as normal.
Healthy, inbred, gender-matched Sprague-Dawley rats weighing 200–250g were used for the study. They were individually housed and maintained on normal food and water ad libitum. The animals were periodically weighed before and after the experiment. The rats were anaesthetised before and during the infliction of the experimental wounds, as described by Morton and Malone.19 The surgical interventions were carried out under sterile con­ditions using ketamine anaesthesia (120mg/kg of body weight). Animals were closely observed for any infection; any with signs of infection were separated, excluded from the study and replaced.
Wound-healing activity
Excision, incision and dead space wound models were used to evaluate the wound-healing activity of M. recutita flower extract.
l Excision wound model?Animals were anaes­thetised before and during the creation of the excision wounds (Fig 1). The dorsal fur of the animals was shaved with an electric clipper and the area on the back of the animals where it was anticipated the wound would be created was outlined with methylene blue using a circular stainless steel stencil. A full-thickness excision wound (circular area 300mm2 and 2mm deep) was created along the markings using toothed forceps, a surgical blade and pointed scissors.
The animals were divided into two groups of six:
l Control group — given a placebo (water)
l Test group — given the flower extract in their drinking water at a dose of 120mg/kg daily until complete epithelialisation occurred. As the average rat consumes 110ml of water/kg/day, we dissolved 120mg of flower extract in 100ml of drinking water. This was done every day, and extra care was taken to avoid variations in the dose.
The wound closure rate was assessed by tracing the wound on days 1, 5, 10 and 15 post-wounding using transparent paper and a permanent marker. Graph paper was used when determining the wound area. Epithelialisation was considered to have occurred when the eschar fell off without leaving a residual raw wound.
l Incision wound model?The dorsal fur was shaved with an electric clipper, and a 6cm longitudinal paravertebral incision was made through the skin and cutaneous muscle on the back.20 Surgical sutures were applied to the parted skin at 1cm intervals. The wounds were left undressed (Fig 1).
The animals were divided into two groups of six:
l Control group — given a placebo (water)
l Test group — given flower extract orally in their drinking water at a dose of 120mg/kg.
The sutures were removed on day 8 and the treatment was continued. The wound-breaking strength was measured on day 10 using the method described by Lee.21 (The incision was expected to take 10 days to heal.)
l Dead space wound model?Dead space wounds (Fig 1) were inflicted by implanting sterile cotton pellets (5mg each), one on either side of the groin and axilla, on the ventral surface of each rat using D’Arcy et al.’s technique, as described by Turner.22
The animals were divided into two groups of six:
l Control group — given a placebo (water)
l Test group — given flower extract orally in their drinking water at a dose of 120mg/kg daily.
On day 10 granulation tissue that had formed on the implanted cotton pellets was carefully removed under anaesthesia, and the weight of the wet granulation tissue was noted. The granulation tissue was dried at 60°C for 12 hours, when the weight of the dry granulation tissue was recorded. To the dried tissue 5ml 6N (normal) hydrogen chloride was added and kept at 110°C for 24 hours. The neutralised acid (hydrolysate) in the dry tissue was used to determine the hydroxyproline content,23 which is an indicator of increased collagen turnover. An additional sample of wet granulation tissue was preserved in 10% formalin for histological studies.
Determination of wound-breaking strength
The anaesthetised animal was secured to the table, and a line was drawn on either side of the incision 3mm away from the suture line. The line on either side of the suture line was gripped using forceps, one at each side opposed to each other. One of the forceps was supported firmly, whereas the other was connected to a freely suspended lightweight measuring jar. Water was added slowly and continuously until the wound started to gape. As soon as this was noticed, the addition of water was stopped. The
volume of water added to the wound was noted
as a measure of breaking strength in grams. Three
readings were recorded for each incision. The mean reading for the group was taken as an individual value of the wound-breaking strength. The mean value gives the breaking strength for a given group.
Estimation of hydroxyproline content
Dry granulation tissue from the dead space wounds from both control and treatment groups was used to estimate the hydroxyproline content. The hydroxyproline content in the neutralised acid (hydrolysate) was subsequently oxidised by sodium peroxide in the presence of copper sulphate. It was then complexed with paradimethylaminobenzaldehyde to develop a pink colour, which was measured at 540nm with a spectrophotometer.
Histological study
Granulation tissue from the dead space wounds was obtained on day 10 from the test and control group animals for histological study. For the better appreciation of collagen deposition, Masson’s trichrome stain was used, which stains the fibres green.
Phytochemical screening methods
l Test for saponins?The M. recutita extract (300mg) was boiled in 5ml water for two minutes. The mixture was then cooled, mixed vigorously and left for three minutes. The formation of froth indicates that saponins are present.24
l Test for tannins?To an aliquot of the extract (dissolved in water), 2ml of 2% sodium chloride was added, filtered and mixed with 5ml 1% gelatin solution. Precipitation indicates that tannins are present.24
l Test for triterpenes?The extract (300mg) was mixed with 5ml chloroform and warmed for 30 minutes. A few drops of concentrated sulphuric acid were added and mixed well. The appearance of a red colo0ur indicates that triterpenes are present.25
l Test for alkaloids?The extract (300mg) was digested with 2M hydrogen chloride, and the acidic filtrate was mixed with amyl alcohol at room temperature. Pink colour of the alcoholic layer indicates that alkaloids are present.25
l Test for flavonoids?The presence of flavonoids was determined by using 1% aluminum chloride solution in methanol, concentrated hydrogen chloride, mag­nesium turnins and potassium hydroxide solution.24
The thin-layer chromatography of the aqueous extract on silica gel was done using the medium chloroform:methanol (9:1 vol/vol) and chloroform: acetone (1:1 vol/vol) as the mobile phase.
Antimicrobial activity
The following organisms were tested: Pseudomonas aeruginosa, beta-haemolytic streptococci, Enterobacter agglomerans, Escherichia coli, Staphylococcus aureus and Cedecea neteri. The bacterial strains were obtained from fresh colonies grown on MacConkey agar and blood agar plates. Sensitivity testing was done using Muller Hinton agar plates. A known volume of bacterial suspension was transferred to each microplate well. Ten microlitres of aqueous extract (5mg/ml) of M. recutita flower was added to the microplate wells and incubated at 35–37°C for
18–20 hours. Results were analysed visually.
Statistical analysis
The mean wound area, epithelialisation period, tensile strength, wet and dry weight and hydroxyproline content of the granulation tissue of the test and control groups were compared using Mann-Whitney U non-parametric tests. Data were analysed using SPSS (Version 12.0, Chicago, US) and the
p value was set at <0.05 for all analyses.
Results
The phytochemical analysis of the flower extract showed that polyphenols, volatile oils and flav­onoids were present, whereas other constituents such as triterpenoids, tannins, alkaloids and carboxylic acids were absent.
A significant increase in wound-healing activity was observed in the animals treated with the
M. recutita extract when compared with the
controls. Table 1 shows the effects of the oral administration of M. recutita extract on wound healing in rats with excisional wounds. In this model, M. recutita-treated animals were found to epithelialise faster (15.20 days ± 0.13) than the controls (18.0 days ± 0.10). The reduction in wound area in the test group was 61% when compared with the controls (48%); this was statistically significant (p<0.002).
In the incision wound model, a significant increase in the wound-breaking strength (654.10 ± 16.50) was observed when compared with the controls (428.30 ± 14.47) (Table 2).
In the dead space wound model the test group showed significantly increased levels of hydroxyproline content (51.80mg/g ± 3.70) compared with the controls (33.30mg/g ± 3.70) (Table 2). Furthermore, a significant increase was observed in the weight of wet and dry granulation tissue in the test animals (p<0.001).
The histological study of the granulation tissue obtained on day 10 from the test animals showed increased well-organised bands of collagen, more fibroblasts and few inflammatory cells (Fig 2) when compared with a section of granulation tissue from the controls, which revealed inflammatory cells and scanty collagen fibres and fibroblasts (Fig 3).
The microbial organisms, including Pseudomonas aeruginosa, beta-haemolytic streptococci, Entero­bacter agglomerans, Escherichia coli and Staphylococcus aureus, were resistant to the extract. The rare microorganism Cedecea neteri was found to be sensitive to the extract.
Discussion
In our study the aqueous flower extract of M. recutita significantly increased the rate of wound contraction and epithelialisation, wound-breaking strength, and the weight and hydroxyproline content of the granulation tissue.
Granulation tissue formed in the final part of the proliferative phase is primarily composed of fibro­blasts, collagen, serous fluid and new small blood vessels. The increased hydroxyproline content in granulation tissue is indicative of an increased collagen turnover. Collagen, the major component of granulation tissue, strengthens and supports extracellular tissue. As the amino acid hydroxyproline is a component of collagen, it has been used as a biochemical marker for tissue collagen.26
The main constituents of the M. recutita flowers include several phenolic compounds, primarily the flavonoids apigenin, quercetin, patuletin, luteolin and their glucosides.27 The principal components of the essential oil extracted from the flowers are the terpenoids alpha-bisabolol and its oxides, and azulenes, including chamazulene.27 Our phytochemical analysis of the flower extract, achieved with qualitative and thin-layer chromatography, showed the absence of active constituents such as triterpenoids, tannins, alkaloids and carboxylic acids and the presence of polyphenols, volatile oils and flavonoids.
M. recutita has been reported to have a controlling effect on menopausal symptoms5 and selective
oestrogen-receptor modifying (SERM) effects.28 Tamoxifen, a SERM agent, delays wound healing but improves scar formation.29 Different SERM agents have different oestrogenic and anti-oestrogenic effects, depending on the tissue and cell type. It is thought the wound-healing properties of M. recutita are caused by its oestrogenic effects on the skin.8,9
While the anti-inflammatory and antipruritic effects of M. recutita are attributed to its possible antihistaminergic and antiserotonergic properties,12 the possibility of glucocorticoid-like activity cannot be ruled out. Glucocorticoids are potent anti-inflammatories and immunosuppressants, and so suppress wound healing.30 However, honey is known for both its anti-inflammatory and wound healing31 and immunostimulating properties.32 Matricaria recutita too may have anti-inflammatory, immunostimulating and healing properties.
The anxiolytic33 and opioid-like16 properties of
M. recutita might enhance wound healing by affecting the hypothalamic-pituitary-adrenal/gonadal axis and potentiating platelet aggregation.18
Matricaria recutita also has been reported to have an inhibitory effect on cytochrome P450 enzymes,34 which might contribute to its healing properties by inhibiting the metabolism of some pro-healing endogenous substances.
Matricaria recutita has been shown to have antioxidant activity in vitro and potent anti-inflammatory action in animal model studies.5 Our phytochemical study demonstrated the presence of flavonoids,
supporting the prescence of antioxidant properties. Inflammation is a prerequisite for wound healing; anti-inflammatory drugs suppress healing. Given its potent anti-inflammatory effect, the way in which M. recutita promotes wound healing needs to be explored. While there has been controversy over the role of free radicals in wound healing,35 it should
be noted that research has showed that the healing pro­perties of honey correlated well with its anti­oxidant properties.36
After analysing our results and previous reports on chamomile, we hypothesise that its wound healing properties are due to its possible antioxidant, oestrogenic, antihistaminergic, antiserotonergic, immunomodulating and opioid-like activities. As M. recutita extract did not inhibit growth of microorganisms that cause wound infection, its healing properties are not related to its antimicrobial activity.
Even though the extract did not contain any of the major phytochemicals, such as triterpenoids, tannins, carboxylic acids and steroids, we cannot rule out the presence of other phytochemicals that promote wound healing.
Conclusion
Our data demonstrate that M. recutita extract facilitates wound healing, which may be due to its
oestrogenic, antioxidant, immunomodulating or some unknown activities. However, this needs to be studied further, to isolate the active ingredients that promote wound healing, before it can be considered for clinical use.
Declaration of interest
None.

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