Karen du Toit1
1Department of Pharmaceutical Chemistry, University of KwaZulu-Natal, Durban, South Africa
2School of Pharmacy and Pharmacology, University of KwaZulu-Natal, Durban, South Africa
3Department of Pharmacology, University of KwaZulu-Natal, Durban, South Africa
Private Bag X54001, Durban 4000, South Africa
Received: 10 May 2010
Accepted: 24 Feb. 2011
Published: 09 May 2011
How to cite this article:
Du Toit K, Kweyama A, Bodenstein J. Anti-inflammatory and antimicrobial profiles of
Scilla nervosa (Burch.) Jessop (Hyacinthaceae). S Afr J Sci. 2011;107(5/6), Art. #259, 5 pages.
© 2011. The Authors. Licensee: OpenJournals Publishing. This work is licensed under the Creative Commons Attribution License.
ISSN: 0038-2353 (print)
ISSN: 1996-7489 (online)
Anti-inflammatory and antimicrobial profiles of Scilla nervosa (Burch.) Jessop (Hyacinthaceae)
In This Research Letter...
• Materials and methods
• Preparation of extracts from S. nervosa
• Assessment of croton oil-induced oedema
• Assessment of microbial susceptibility
• Data analysis
• Results and discussion
• Reduction of croton oil-induced oedema
• Antimicrobial activity
• Methanol–dichloromethane extract
• Methanol–ethanol extract
Scilla nervosa (Burch.) Jessop (Hyacinthaceae) [=Schizocarphus nervosus (Burch.) Van der Merwe]
is a well-known plant in traditional medicine in South Africa, used for conditions associated with pain and
inflammation, such as rheumatic fever. However, the topical anti-inflammatory and antimicrobial activities
of the plant have not been investigated. A bioassay-guided fractionation approach was implemented to
determine the biological activities of different extracts. A crude methanol extract was prepared from
the bulbs to investigate the anti-inflammatory properties in a mouse model of acute croton oil-induced
auricular contact dermatitis. The non-polar and polar components present in the methanol extract were
separated by extraction with dichloromethane and ethanol, respectively; and their antimicrobial activity
against the invasive pathogenic microorganisms Staphylococcus aureus, Klebsiellla pneumoniae
and Candida albicans was investigated using a microplate method. Oedema induced by application
of croton oil was significantly reduced 3 h (~66%) and 6 h (~40%) after treatment with the extracts.
Anti-inflammatory activity was ~1.8-fold lower at 6 h, suggesting a potent, short-acting effect. The
non-polar extract exhibited greater efficacy and potency against the microorganisms than the polar
extract. The non-polar extract was equipotent against S. aureus and K. pneumoniae, but
twice as potent against C. albicans as against the bacteria, suggesting little discrimination
between Gram-positive and Gram-negative bacteria but specificity for the fungal yeast. The polar
extract was the least potent against K. pneumoniae, but 10-fold more potent against C. albicans,
suggesting specificity for Gram-positive bacteria and the fungal yeast. S. nervosa contains compounds
that are individually, or in combination, potent anti-inflammatory and antimicrobial agents. The anti-inflammatory
activity demonstrated here may rationalise the use of the plant in traditional medicine.
The skin is a major barrier that protects the body against trauma and invasion by microbial
pathogens. To protect the body from the external environment, the skin has many defence
mechanisms that are normally closely regulated to suppress inappropriate activity (e.g. psoriasis)
and to destroy bacterial (e.g. impetigo and acne) and fungal (e.g. mucocutaneous candidiasis) infections.1
Skin disorders are commonly treated with a combination of antibiotics, corticosteroids, antihistamines
and moisturisers to reduce itching, infection and inflammation. However, chronic treatment with corticosteroids
suppresses lymphocyte function and decreases collagen synthesis, causing skin atrophy and increasing the risk
of opportunistic infections. In addition, opportunistic infections may be difficult to treat as a result of
acquired resistance against current antimicrobial therapies.2 Therefore, new therapeutic approaches
are being sought, in which not only the inflammatory response is effectively suppressed, but also the causative
Scilla nervosa (S. nervosa) (Burch.) Jessop (Hyacinthaceae) or Wild squill is also known as
Schizocarphus nervosus (Burch.) Van der Merwe, in isiZulu as Ingcino and in Afrikaans as Blouberglelie.
It is indigenous to Botswana but has naturalised in the grasslands of southern Africa.3 Bulbs are used in
traditional medicine by the Tswana people to treat infertility in women, by the Sotho people to treat
gall sickness in livestock4 and constipation,5 dysentery and nervous conditions
in children,6 by the Zulu people to treat pain in rheumatic fever, which primarily affects
children between the ages of 5 and 15,5 and by the Xhosa people to treat dysentery and
nervous conditions in children.3 At least one traditional use indicates possible anti-inflammatory
properties of the bulbs: treatment of pain in rheumatic fever. However, there have been no reports on the topical
anti-inflammatory activity in vivo to rationalise its traditional use and antimicrobial activity
in vitro to probe additional medicinal properties. We therefore investigated these activities.
Acetone, ethanol, dichloromethane and methanol were obtained from Merck (Darmstadt, Germany).
Amphotericin B, neomycin solution, croton oil, diclofenac and dimethylsulphoxide were obtained
from Sigma (St Louis, MO, USA). p-Iodonitrotetrazolium chloride violet, Mueller-Hinton
broth and Sabouraud dextrose broth were obtained from Fluka (Buchs, Switzerland).
Preparation of extracts from S. nervosa
S. nervosa was bought in June 2008 from the Fig Tree Indigenous Nursery (Westville, Durban,
South Africa). The plants were identified and a voucher (Du Toit2, NU) was lodged at the University
of KwaZulu-Natal Herbarium (Pietermaritzburg, South Africa). All of the plants were of the same
developmental stage. The fresh bulbs (1.5 kg) were chopped into small pieces and dried overnight. A
bioassay-guided fractionation approach was followed to investigate the biological activities of
different extracts. Pieces were soaked in methanol for 3 days, filtered and dried in a rotary
evaporator (Heidolph, Darmstadt, Germany). The crude methanol extract (42.3 g), containing polar
and non-polar components, was used for the anti-inflammatory assays and to prepare two additional
extracts for the antimicrobial assays. Two amounts of 1 g of the crude methanol extract were
sonicated for 1 h in equal volumes (20 mL) of dichloromethane or ethanol to separate the non-polar
and polar components, respectively. The two extracts were filtered and dried in the evaporator to
yield a methanol-dichloromethane and methanol-ethanol extract. Extracts were stored at -80 °C until further use.
Assessment of croton oil-induced oedema
Ethical approval (058/09/Animal) from the University of KwaZulu-Natal Animal Ethics Subcommittee
was obtained prior to the investigation of croton oil-induced oedema7 in a mouse model.
Guidelines by the University of KwaZulu-Natal Animal Ethics Subcommittee and Biomedical Resources
Unit for the maintenance and treatment of laboratory animals were followed.
This topical study design was based on previous studies where the inhibition of croton oil-induced
auricular oedema by emu oil was investigated to screen for agents against contact
dermatitis8,9 and to assess the anti-inflammatory profiles of flavonoids.10
Briefly, 20 8-week-old male Balb/c mice, weighing approximately 30 g each, were used. The mice
were divided into two groups of 10 mice each (two per condition). One group was examined 3 h
after treatment and the other group 6 h after treatment. Equal volumes of croton oil and
acetone (as a vehicle) were mixed and 50 ml of the mixture was applied to the inner surface
of the right auricle of each mouse to induce oedema. The left ears were untreated.
Acetone has not been documented to have an anti-inflammatory effect by itself.1,11
After 1 h, different amounts of the crude methanol extract in acetone were applied
(0 mg (control)), 0.1 mg, 0.5 mg and 1 mg in 50 mL) to the right auricle (~1 cm2)
and any reduction in oedema was assessed 3 h and 6 h after extract application.
Mice were euthanised after 3 h or 6 h, and left and right auricle biopsy specimens were
obtained using a 6-mm biopsy punch. The specimens were weighed to assess any differences in oedema.
The non-steroidal anti-inflammatory drug diclofenac in acetone (0.1 mg in 50 ml) was included as a positive control.
Assessment of microbial susceptibility
Mueller-Hinton broth was inoculated with Staphylococcus aureus or Klebsiella pneumoniae
and grown in an incubator at 37 °C, with an optical density of 0.8 at 490 nm. Sabouraud dextrose broth
was inoculated with Candida albicans and grown in an incubator at 37 °C, with an optical density
of 0.5 at 600 nm. S. aureus (strain 12600), K. pneumoniae (strain 13883) and C.
albicans (strain 10231) cultures were obtained from American Type Culture Collection (ATCC)
(Manassas, VA, USA). The broths were prepared according to the manufacturer’s protocol.
The microbial susceptibility assay was based on a microplate method10,12 but with modifications.
The two extracts were prepared in pure dimethylsulphoxide at concentrations of 0.01 mg/mL, 0.05 mg/mL,
0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL and 3 mg/mL. Firstly, 100 ml/well of sterile broth was added to a clear, sterile 96-well
microtitre plate (Corning Life Sciences, Acton, MA, USA). Secondly, 6 mL/well of the extract was added
and the plate tapped. Thirdly, 94 mL/well of sterile water was added and the plate tapped. Finally, 100
mL/well of the appropriate microbial culture was added and the plate was tapped to mix the contents and
then incubated at 37 °C for 18 h. Therefore, with a final volume of 300 mL/well and a dilution factor of
50x, the final concentration of dimethylsulphoxide/well was 2% v/v and the final concentrations of the
extracts/well were 0.2 mg/mL, 1 mg/mL, 2 mg/mL, 10 mg/mL, 20 mg/mL, 40 mg/mL and 60 mg/mL. Growth of the
microorganisms was not significantly inhibited by the 2% v/v dimethylsulphoxide (data not shown).
The positive controls were neomycin against S. aureus and K. pneumoniae, and amphotericin
B against C. albicans (100 mL at a concentration of 300 mM was added in step 2 above,
therefore with a final volume of 300 mL/well and a dilution factor of 3x, the final concentrations
were 100 mM). For S. aureus and K. pneumoniae, 40 mL/well of p-iodonitrotetrazolium
chloride violet (400 mg/mL in water) was added and the plate tapped and incubated at 37 °C for
15 min. Microbial growth was quantified by colourimetry (490 nm) for bacteria and optical density
(600 nm) for C. albicans in a microplate reader (BioTek ELx800, Winooski, VT, USA).
Data are reported as the mean ± standard error of the mean of five (anti-inflammatory assays) and
four to six (antimicrobial assays) independent experiments with duplicate measurements per condition.
For the anti-inflammatory assays, two mice per each condition (representing the duplicate measurement)
were used. Oedema was quantified by calculating the difference in mass of the right and left auricle
biopsy specimens and expressed as a percentage of the croton oil control. For the antimicrobial assays,
microbial growth was quantified as a percentage of the control without any plant extract. Two wells
per each condition (representing the duplicate measurement) were used. GraphPad Prism13
was used to determine the IC50 value (the concentration of an inhibitor at which a response
is reduced by 50%) through non-linear least squares regression. Minimum inhibitory concentrations (MICs)
were deduced from the graphs. Statistical comparisons were made by one-way analysis of variance (ANOVA)
followed by Bonferroni’s post-test or by Student’s two-tailed paired t-test to
determine p-values. A value of p < 0.05 was considered significant.
Reduction of croton oil-induced oedema
The anti-inflammatory activity of S. nervosa extract in a mouse model of acute croton
oil-induced oedema has not been previously studied. Increased skin thickening as a result of
oedema is often the first sign of skin irritation and local inflammation. Oedema is one indicator
of the complex processes that occur during skin inflammation, including increased vascular
permeability, swelling within the dermis and proliferation of epidermal keratinocytes. Ear
oedema was measured in the auricular skin at 3 h and 6 h following treatment with the extract
to determine sustained anti-inflammatory activity. Topical application of the crude methanol
extract in acetone at three different doses significantly inhibited the croton oil-induced
increase in skin weight after 3 h (~3-fold) and 6 h (~1.8-fold), indicating the therapeutic
ability of S. nervosa to ameliorate croton
oil-induced contact dermatitis (Figure 1). This was supported by observed reductions in skin
thickness, weight and redness. The greatest anti-inflammatory effects were observed with 0.5
mg at 3 h (inhibition of oedema was 65.5 ± 7.0%) and 0.1 mg at 6 h treatment (inhibition of
oedema was 46.2 ± 4.1%). Anti-inflammatory effects of each dose of the extract were significantly
lower (~1.8-fold) at 6 h post-treatment than at 3 h, suggesting that S. nervosa is an
anti-inflammatory agent with a relatively short duration of action, similar to that of the
diclofenac control. Diclofenac at 0.1 mg significantly inhibited oedema after 3 h (64.8 ± 5.4%)
and 6 h (43.9 ± 4.1%) treatment. No mice died during the study, nor were any adverse effects
observed with the different doses of extract. These outcomes suggest that S. nervosa
was non-toxic in mice up to a topical dose of 1 mg/cm2.
FIGURE 1: Anti-inflammatory activity, shown as a percentage of oedema (mean ± s.e.m.), of Scilla nervosa extract, diclofenac and the control (no treatment).
The mechanism of the observed anti-inflammatory activity is unknown. It would be worthwhile
to determine the expression of cytokines, such as interleukins (IL) and tumour necrosis
factor (TNF), as well as myeloperoxidase and cyclo-oxygenase activities. In a study on the
anti-inflammatory effects of an Asparagus cochinchinensis extract, Lee and co-workers1
demonstrated a significant reduction in mice ear thickness and weight 6 h after treatment. Although the
components of the ethanolic extract were unknown, mechanistic studies indicated a reduction in the levels of
IL-1β, TNF-α and myeloperoxidase activity. A study by Oliveira de Melo and co-workers14
on different extracts of Nectandra falcifolia also demonstrated reduced myeloperoxidase activity.
Microbial skin infections often cause local inflammation as part of the immune response. Because it
is demonstrated here that bulbs of S. nervosa possess anti-inflammatory activity, the
susceptibility of the important invasive Gram-positive S. aureus, Gram-negative K.
pneumoniae and the fungal yeast C. albicans to two different extracts prepared
from S. nervosa was subsequently assessed. These microorganisms are implicated in skin
conditions such as boils, impetigo, acne, mucocutaneous candidiasis and opportunistic infections
in immunocompromised patients.
Plant extracts prepared from different solvents contain different ingredients in different ratios.
We therefore prepared two extracts: a methanol-dichloromethane extract and a methanol-ethanol
extract. The positive control neomycin, at 100 mM, significantly inhibited the growth of S.
aureus (83.8 ± 0.6%) (Figure 2a and 2d) and K. pneumoniae (87.2 ± 2.1%) (Figure 2b
and 2e), whilst amphotericin B, at 100 mM, significantly inhibited the growth of C. albicans
(95.8 ± 0.8%) (Figure 2c and 2f).
FIGURE 2: Antimicrobial activity, shown as a percentage of growth (mean ± s.e.m.) of Scilla nervosa extracts, methanol-dichloromethane (a–c) and methanol-ethanol (d–f), against the invasive pathogenic microorganisms Staphylococcus aureus (a & d), Klebsiella pneumoniae (b & e) and Candida albicans (c & f).
Growth of S. aureus (Figure 2a), K. pneumoniae (Figure 2b) and C. albicans (Figure 2c) was
inhibited in a dose-dependent manner after treatment with the methanol–dichloromethane extract; and total
suppression was reached at higher concentrations, suggesting bactericidal and fungicidal activity. The average
MIC values were calculated as 40 mg/mL, 60 mg/mL and 40 mg/mL and the average IC50 values
were calculated as 1.8 ± 0.6 mg/mL, 2.0 ± 0.4 mg/mL and 1.0 ± 0.4 mg/mL for S. aureus, K.
pneumoniae and C. albicans, respectively. This non-polar extract showed equivalent activities
against S. aureus and C. albicans and was the least active against K. pneumoniae.
However, it was almost twice as potent against C. albicans (IC50 ratio of ~2) when
compared to S. aureus and K. pneumoniae, which exhibited similar potencies.
Growth of S. aureus (Figure 2d), K. pneumoniae (Figure 2e) and C. albicans
(Figure 2f) was inhibited in a dose-dependent manner after treatment with the methanol–ethanol
extract. Average MIC values could not be deduced because total growth suppression could not be
reached even at higher doses (data not shown), suggesting bacteriostatic and fungistatic activity.
The general trend observed was that the polar extract was the least active against K. pneumoniae
and the most active against C. albicans. The average IC50 values were calculated as
6.2 ± 3.6 mg/mL, 16.2 ± 4.0 mg/mL and 1.6 ± 0.4 mg/mL for S. aureus, K. pneumoniae
and C. albicans, respectively. This polar extract was almost 10-fold more potent against C.
albicans when compared to K. pneumoniae (IC50 ratio of ~10) and almost 4-fold
more potent when compared to S. aureus (IC50 ratio of ~3.9). Furthermore, it was
more than 2.5-fold more potent against S. aureus than K. pneumoniae, against which
it was the least potent (IC50 ratio of ~2.6).
These findings suggest that there may be little difference in activity of the non-polar
extract against Gram-positive and Gram-negative bacterial species. The polar extract
exhibited specificity towards Gram-positive bacterial species; both extracts exhibited
specificity towards the fungal yeast species, but the non-polar extract was the more
potent and had the higher efficacy of the two extracts. These extracts contain different
compounds (homoisoflavanones and/or stilbenes) in different ratios.5 It is
possible for these compounds to potentiate each other’s activities at different
concentrations to dictate microbicidal or microbiostatic activity. In a review by Van
Vuuren15 on the antimicrobial activity of South African medicinal plants, it
was suggested that the identification of a single active component responsible for
activity is becoming more improbable and that research should be focusing on the
investigation of a combination of compounds to achieve greater efficacy. Based on
our findings, it is clear that the non-polar extract was more potent against the
microorganisms than the polar extract. Future aims are to determine the specific
compounds present in each extract as well as their concentrations, and to determine
individual activity as well as their combined activity.
Our findings suggest that extracts from S. nervosa bulbs may be important in the treatment
of not only idiopathic inflammatory skin diseases, but also bacterial and fungal skin diseases.
Furthermore, our findings rationalise the traditional use of the plant as an anti-inflammatory
agent and may be useful towards the development of new anti-inflammatory and antimicrobial therapies
with specificity towards certain microbes. Finally, it will be worthwhile to characterise the
various mechanisms of the anti-inflammatory activity.
We thank Ms Sithabile Buthelezi for experimental assistance and Dr Christina Potgieter
at the Bews Herbarium, University of KwaZulu-Natal for current information on the plant
name. This study was funded by a University of KwaZulu-Natal Competitive Research Grant.
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