WO2009077850A1

WO2009077850A1 - Antimicrobial composition comprising an extract from cyclopia

Info

Publication number: WO2009077850A1
Application number: PCT/IB2008/003539
Authority: WO
Inventors: Marinda Bloom; Elizabeth Joubert
Original assignee: Stellenbosch University; Arc Infruitec-Nietboorbij
Priority date: 2007-12-18
Filing date: 2008-12-18
Publication date: 2009-06-25
Also published as: ZA201003568B

Abstract

An antimicrobial composition comprising an extract from a plant belonging to the genus Cyclopia is produced by preparing an extract at a concentration in the range of 0.5 mg/ml to 800 mg/ml from the leaves of Cyclopia subternata or Cyclopia genistoides. An effective amount of the antimicrobial composition of the invention is then applied to a substrate or surface for use in a method of preventing or treating a microbial infection or contamination.

Description

ANTIMICROBIAL COMPOSITION COMPRISING AN EXTRACT FROM

CYCLOPIA

FIELD OF THE INVENTION

The invention relates to an antimicrobial composition comprising a plant extract and to a method of preventing or treating a microbial infection or contamination.

BACKGROUND TO THE INVENTION

Many of the commonly used food preservatives and microbial agents are synthetic compounds. In recent years, there has been an increased interest in avoiding or eliminating the use of synthetic compounds and in developing and promoting the use of natural materials for use as antimicrobial agents both in food preservation and in agricultural practices. This is due not only to the emergence of antimicrobial drug resistance in human pathogens and food spoilage organisms as a result of increased use of synthetic antibiotics and antimicrobial agents and compositions, but also to a general increased interest in the use of natural materials. Various plant extracts, such as those from green tea, rooibos tea and oolong tea, have been researched for their anti-microbial properties with varying degrees of success.

Preservation of food from microbial contamination is necessary as food borne illnesses still occur at a high frequency. Bacteria are generally the most important cause of food borne diseases. For example, Staphylococcus aureus is a common Gram-positive bacterium that causes food poisoning.

Escherichia coli is a faecal bacterium and enterohemmorhagic strains thereof may cause food poisoning. Pseudomonas aeruginosa is a virulent opportunistic pathogen in humans that may cause devastating acute infections, including pneumonia and sepsis. Multi-drug resistant pathogenic bacteria include P. aeruginosa, S. aureus and E. coli. Propionibacterium acnes, which causes acne, have also developed resistance against a number of antibiotics. The yeast Candida albicans is an example of a multi-drug resistant human fungal pathogen, which is responsible for many clinical yeast infections especially in immunocompromised individuals.

There is therefore a need for an improved antimicrobial composition that may be used as a natural preservative in food preservation or in agriculture.

OBJECT OF THE INVENTION

It is an object of this invention to provide an alternative antimicrobial composition that may alleviate, at least to some extent, the abovementioned problems.

SUMMARY OF THE INVENTION

In accordance with the invention there is provided an antimicrobial composition comprising an extract from at least one plant belonging to the genus Cyclopia.

Further features of the invention provide for the plant to be either or both Cyclopia subternata and Cyclopia genistoides; for the extract to be extracted from the leaves and stems of the plant; for a solvent to be used to perform the extraction, for the solvent to be selected from the group consisting of water, ethanol, and ethyl acetate; for the concentration of the extract to be in the range of 0.5 mg/ml to 800 mg/ml preferably in the range of 10 mg/ml to 50 mg/ml; for the extract to be used in combination with another extract from a plant, such as Aspalathus linearis, in a ratio in the range of 2:3 to 3:2.

The invention also provides for a method of inhibiting the growth of at least one microbe selected from the group consisting of Escherichia coli; Salmonella enterica; Vibrio spp.; Staphylococcus aureus; Pseudomonas aeruginosa; Propionibacterium acnes; Enterococcus feacalis; Streptococcus mutans; Listeria monocytogenes; and Candida albicans, the method including contacting the microbe with an antimicrobial composition comprising an extract from at least one plant belonging to the genus Cyclopia.

The invention further provides a method of preventing or treating a microbial infection or contamination, the method comprising applying to a substrate or a surface an effective amount of the antimicrobial composition of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described, by way of example only, with reference to the accompanying figures in which

Figure 1 shows a table of the bacterial strains used in Example 1 to evaluate the antimicrobial activity of the tea extracts and the corresponding growth media in which the bacteria were maintained; Figure 2 shows a table of the inhibitory effect of the tea extracts on viable bacterial cell numbers as described in Example 1 , in which "nd" means not determined;

Figure 3 shows a table of the inhibitory effect of the tea extracts on the turbidity of bacterial cultures as described in Example 1 ;

Figure 4 shows a graph of the effect of (■) 10 mg/ml A. linearis (A) C. subternata and (•) C. genistoides extracts on viable E. coli cell numbers, when compared to PD medium only (O) as described in Example 2;

Figure 5 shows a table of the inhibitory effect of the tea extracts on viable C. albicans cell numbers as described in Example 3; and

Figure 6 shows a table of the antimicrobial activity (% inhibition) of the organic fractions of Raps unfermented rooibos and Raps C. subternata against S. aureus and L monocytogenes as described in Example 4.

DETAILED DESCRIPTION WITH REFERENCE TO DRAWINGS

The antimicrobial composition comprising an extract from a plant belonging to the genus Cyclopia is produced by preparing an extract at a concentration in the range of 0.5 mg/ml to 800 mg/ml from the leaves of Cyclopia subternata or Cyclopia genistoides. Typically, a solvent is used to perform the extraction. Preferably the solvent is water, 70% ethanol, or ethyl acetate, and the extract is prepared according to procedures outlined in PCT Application number WO2006081989 or Joubert et al. (2004). Briefly, the extracts are prepared from plant material harvested and dried in a forced-air drying tunnel without shredding prior to drying to prevent oxidative changes. The plant material is then pulverized with a Retch mill with a sieve and stored in sealed plastic containers at room temperature in the dark until used. Solvent is then added to the extract to produce the antimicrobial composition. The antimicrobial composition demonstrates high inhibitory activity against many microbes, including Escherichia coli; Salmonella enterica; Vibrio spp.; Staphylococcus aureus; Pseudomonas aeruginosa; Propionibacterium acnes; Enterococcus feacalis; Streptococcus mutans; Listeria monocytogenes; and Candida albicans.

It is also proposed that the extract is combined with a similar extract from Aspalathus linearis in a ratio of 1 :1. An effective amount of the antimicrobial composition of the invention is then applied to a substrate or a surface for use in a method of preventing or treating a microbial infection or contamination.

EXAMPLE 1 : Antibacterial activity of extracts from Cyclopia spp.

Materials and Methods

Plant material and reagents

Dried extracts prepared from Cyclopia subternata were supplied by Raps GmbH & Co. (Germany) (Prod. No. 4448002; manufacturing date: 31/03/2005, prepared according to WO2006081989). Cyclopia genistoides extract was supplied by the Post-Harvest & Wine Technology Division of ARC Infruitec-Nietvoorbij (Stellenbosch, South Africa). The dried extracts were prepared by adding 250 ml dichloromethane to 50 g plant material and stirring the suspension for 60 minutes. The suspension was then filtered through Whatman filter paper (number 4). This filtering step was repeated four times and the last filtrate left to stand overnight. The filtrate was then filtered once more as before. To the residual plant material obtained was added 250 ml 80 % ethanol, and the suspension stirred for 60 minutes. The suspension was then filtered as before and the solvent evaporated under vacuum. The resulting dried extract powder obtained was freeze-dried.

Stock solutions of tea extracts were prepared by dissolving the extract powder in sterile distilled water or 70% ethanol at a concentration of 1 g/ml, followed by addition to the respective media to yield final concentrations of

0.5 mg/ml to 800 mg/ml. The resulting extracts formed tea extracts to be tested for antimicrobial effectiveness. All chemicals were of standard or analytical grade and obtained from BDH Chemicals Ltd (Poole, England); Fluka AG (Buchs, Switzerland); Merck (Darmstadt, Germany) or Sigma

Chemical Co. (St. Louis, USA).

Bacterial cultures

Bacteria were maintained according to standard procedures. Bacterial strains and their respective media used in this study are listed in Figure 1. 10 ml overnight cultures in the appropriate media indicated in Figure 1 were prepared from a 30 μl innoculum of a 80% glycerol stock and incubated at 37⁰C overnight. The overnight cultures were also diluted ten-fold in dH₂O, of which 1 ml was used to prepare pour plates together with 10 ml of the corresponding semi-solid media (1% agar).

Investigation of suspension culture growth inhibition of bacteria in the presence of tea extracts

Inhibition of cell growth as measured by viable plate counts

50 ml test cultures were inoculated in triplicate from overnight cultures to a final concentration of 10⁵ to 10⁶ cells/ml in appropriate media. Tea extracts were added to test cultures at 10 or 100 mg/ml. No extract was added to control cultures. Bacterial cultures were incubated at 37°C in the dark to minimise oxidation under constant agitation (100 rpm). Samples were taken from cultures at Oh, 6h, 12h, 24h and 48h and viable cell counts obtained from duplicate dilution plate counts. Percentage inhibition was expressed as I = [(N_c-N_t)/N_c] x 100, where N₀ and N_t represent the viable cell numbers for the control and treatments, respectively.

Inhibition of cell growth as measured by turbidity

The antimicrobial activity was determined by monitoring the growth of the respective organisms in the presence of 50 mg/ml to 100 mg/ml tea extracts using 96-well microtitre plates (Merck, Germany). Each well was inoculated with 5 μ\ of an overnight culture. The microtitre plates were incubated at 37⁰C under static conditions, with microbial growth measured spectrophotometerically at 600 nm for up to 6 hours. The control wells contained the respective growth media without tea extract, or growth media with tea extract and no inoculum to correct for absorbance by tea components. All samples were tested in triplicate and the standard deviation calculated. Percentage inhibition was expressed as I = [(N_c-N_t)/N_c] x 100, where N_c and N₁ represent the viable cell numbers for the control and treatments, respectively.

Results

Inhibitory effect of the tea extracts on viable bacterial cell numbers

When treated with 10 mg/ml of the tea extracts, 71 % to 97% inhibition of cell growth was observed after 6 hours for all the bacterial test strains, relative to the controls that received no extract (Figure 2). After 48 hrs, inhibition of S. aureus was reduced to 40%-50%, whereas 91 %-96% inhibition of P. acnes and 95%-99% P. aeruginosa was still observed. Treatment with 5 mg/ml tea extracts resulted in 59%-81% inhibition after 6 hrs, with little impact beyond 24 hours (data not shown). Inhibition of bacterial growth as measured by turbidity

When treated with 100 mg/ml of the tea extracts for 6 hours, growth of the bacterial strains indicated in Figure 3 was inhibited by 21%-92% by C. genistoides, and 77%-95% by C. subternata extracts. Treatment with 50 mg/ml extracts resulted in 14%-77% inhibition by C. genistoides, and 53%- 100% inhibition by C. subternata extracts.

60% inhibition (IC₆o) was achieved by 100 mg/ml of the Cyclopia extracts against all the test organisms (except for C. genistoides against S. enterica).

The S. aureus and Ent. feacalis strains seem to be the most sensitive to the extracts as determined by viable cell counts and turbidity measurements.

However, the inhibitory effect of the tea extracts decreased after 48 hours, suggesting a bacteriostatic effect with 6 hrs as optimal for the inhibition of cell growth.

EXAMPLE 2: Comparative antibacterial activity of Cyclopia spp. and A. linearis extracts against E. coli

Materials and methods

Plant material and reagents

Dried extracts prepared from rooibos (A linearis) and honeybush (Cyclopia subternata) tea were supplied by Raps GmbH & Co. (Germany), while the Cyclopia genistoides tea extract was supplied by the Post-Harvest & Wine Technology Division of ARC Infruitec-Nietvoorbij (Stellenbosch, South Africa). The extracts were prepared as described in Example 1. Strains and culture conditions

Escherichia coli DH5σ was cultured overnight at 37⁰C in Luria Bertani (LB) medium under constant agitation (100 rpm).

Effect of tea extracts on cell growth in liquid cultures

Overnight pre-cultures were used to inoculate triplicate sets of 50 ml LB medium (E. coli) at a final concentration of 10⁵ to 10⁶ cells/ml. Extracts from A. linearis and Cyclopia spp. were added at 10 or 100 mg/ml, with the controls not receiving any extract. Bacterial cultures were kept at 37°C protected from light to minimise oxidation of the tea extracts. Duplicate samples were taken at Oh, 6h, 12h, 24h and 48h and used to quantify viable bacterial cells by means of duplicate dilution plate counts. Percentage inhibition was expressed as I = [(N_c-N_t)/N_c] x 100, where N_c and N_t represent the viable cell numbers for the control and treatments, respectively.

Results

The efficacy of the tea extracts used in the experiments was validated using E. coli as a benchmark. In liquid cultures, 10 mg/ml of the A. linearis, C. genistoides and C. subternata extracts inhibited growth of E. coli cells by 60%, 80 and 85% respectively after 6 hrs, when expressed relative to the control (Figure 4). After 24 hrs, 25% and 50% inhibition by A. linearis and both Cyclopia spp. was observed, but the effect diminished thereafter. This is in line with Schepers (2001 ) who reported a 35% cell growth inhibition in E. coli after 12 hrs in the presence of a 5.0 g/L green rooibos tea extract. The extracts from Cyclopia spp. therefore exhibited a greater inhibition against E. coli than did the extract from A. linearis at 10mg/ml. EXAMPLE 3: Antimicrobial activity of Cyclopia spp. extracts against C. albicans

Materials and Methods

Yeast culture and investigation of suspension culture growth inhibition of yeast in the presence of tea extracts

Candida albicans, a human isolate obtained from Dept. Microbiology Stellenbosch, was maintained in Yeast Peptone Dextrose by periodic transfer to agar plates and incubation overnight at 37⁰C. Viable C. albicans cells were determined as described for E. coli cultures in Example 1.

Results

The growth of C. albicans as determined by viable cell numbers was inhibited by 65% by 5 mg/ml of the C. subternata extracts after 6 hours as compared to the 53% inhibition demonstrated by the A. linearis extract (Figure 5). Furthermore, the C. subternata extract demonstrated a longer lasting inhibition in that 50% of C. albicans cell growth was inhibited by 10 mg/ml extract after 48 hours as compared to the 25% inhibition demonstrated by the A. linearis extract at the same time point.

EXAMPLE 4: Antibacterial activity of combined C. subternata and A. linearis extracts

Materials and methods

Fractionation

Extracts of rooibos (Aspalathus linearis) and honeybush (Cyclopia subternata) were fractionated using liquid-liquid partitioning between water and ethyl acetate. The extracts (10 g) were suspended/dissolved in 250 ml deionised water, whereafter they were extracted with 6 x 125 ml portions of ethyl acetate by shake-up method in a separating funnel. The ethyl acetate fractions representing the organic fraction were pooled before rotary evaporation (Bϋchi) and freeze-drying (Virtis Advantage). The remaining water fraction was also subjected to rotary evaporation to remove traces of organic solvent before freeze-drying (Joubert et al, 2004).

Antimicrobial activity testing

The antibacterial activity of the extracts and fractions was determined by monitoring the growth of L. monocytogenes in the presence of 5 mg/ml (final concentration) tea extracts/fractions using U-shaped 96-well microtitre plates. Tea extracts/fractions were dissolved in brain heart infusion broth (BHI) and inoculated with 2% (v/v) of overnight L. monocytogenes cultures which is equivalent to McFarland Standard 2 (6 * 10⁸ cells/ml) (Schepers, 2001 ). As control, L. monocytogenes was grown in BHI broth without added tea extracts/fractions. The microtitre plates were incubated at 37 ⁰C under static conditions for the duration of the growth assay. At one hour intervals, samples were taken and serial dilutions (10^'1 to 10^"8) were made in sterile saline solution (0.85% (w/v) NaCI). The dilution series was then plated onto BHI agar plates and incubated overnight at 37 ⁰C. The number of colonies were enumerated and the % inhibition calculated as (Vc - Vt) ÷ Vc * 100, where Vc is the number of viable cells in the control sample and Vt the number of viable cells in the treated sample. All experiments were done in triplicate with the % inhibition expressed as the mean values obtained. Determining the minimal inhibitory concentrations of the organic fractions

The viability of L monocytogenes in the presence of different concentrations (0.1 to 5 mg/ml) of the organic fractions of the unfermented rooibos extract was determined using the method described for antimicrobial activity testing.

Assessing the antimicrobial activity of combined rooibos and honeybush extracts

The antimicrobial activity of the organic fractions of the unfermented rooibos and honeybush extract was tested at 5mg/ml and 10 mg/ml against S. aureus and L. monocytogenes. The inhibitory effect and possible synergy of the combined fractions at a final concentration of 5 mg/ml and 10 mg/ml were monitored for 48 hours using the method previously described.

Results

When the organic fractions of unfermented rooibos and honeybush were combined in a 1 :1 ratio (5 and 10 mg/ml final concentrations, respectively), inhibition of both S. aureus was similar or higher than when the fractions were used individually (Figure 6). An inhibition of more than 99% was found for both 5 mg/ml and 10 mg/ml after 48 hrs against S. aureus. Notably, the honeybush extract at 5 mg/ml was found to be more effective than the rooibos extract against L monocytogenes, demonstrating an inhibition of

87.9% as compared the 68.2% inhibition demonstrated by the rooibos extract. At 10 mg/ml, the honeybush extract also showed a greater inihibitory activity against L monocytogenes of 99.4% as compared to the 80.5% inhibition shown by the rooibos extract. The combination of honeybush and rooibos extracts at 5 mg/ml demonstrated greater inhibition of S. aureus and

L. monocytogenes, that of rooibos alone, 80.8% as compared to 68.2%. The inhibition of L. monocytogenes by the combination of the two extracts at 10 mg/ml was similar to that obtained for the rooibos extract alone.

For L. monocytogenes, the inhibition by the 5 mg/ml rooibos fraction declined at 24 hrs, but then improved towards 48 hrs. A less significant decline and recovery was also evident for the S. aureus strain against the 5 mg/ml rooibos fraction. The reason for this remains unclear at present, but may be linked to the general growth phase of the organism.

REFERENCES

Joubert, E., Winterton, P., Britz, TJ. , Ferreira, D., 2004. Superoxide anion and α; σ-diphenyl-/?-picrylhydrazyl radical scavenging capacity of rooibos (Aspalathus linearis) aqueous extracts, crude phenolic fractions, tannin and flavonoids, Food Research International 37 : 133-138

Joubert, E., Gelderblom, W.C.A., Louw, A., and de Beer, D., 2008. South African herbal teas: Aspalathus linearis, Cyclopia spp. and Athrixia phylicoides — A review, Journal of Ethnopharmacology, 119(3): 376-412

Scheepers, S., 2001. Anti-microbial activity of rooibos tea (Aspalathus linearis) on food spoilage organisms and potential pathogens. MSc (Food Science) thesis, Stellenbosch University, Stellenbosch, South Africa.

Claims

CLAIMS:

1. An antimicrobial composition comprising an extract from at least one plant belonging to the genus Cyclopia.

2. The antimicrobial composition according to claim 1 , wherein the at least one plant is Cyclopia subternata or Cyclopia genistoides.

3. The antimicrobial composition according to either claim 1 or claim 2, wherein the extract is produced from the leaves and stems of the plant.

4. The antimicrobial composition according to any one of the preceding claims, wherein the concentration of the extract is in the range of 0.5 mg/ml to 800 mg/ml.

5. The antimicrobial composition according to claim 3, wherein the concentration of the extract is in the range of 10 mg/ml to 50 mg/ml.

6. The antimicrobial composition according to any one of the preceding claims, wherein the extract is used in combination with a second extract from another plant.

7. The antimicrobial composition according to claim 6, wherein the second extract is from Aspalathus linearis and is present in a ratio in the range of 2:3 to 3:2.

8. A method of inhibiting the growth of at least one microbe selected from the group consisting of Escherichia coli; Salmonella enterica; Vibrio spp.; Staphylococcus aureus; Pseudomonas aeruginosa; Propionibacterium acnes; Enterococcus feacalis; Streptococcus mutans; Listeria monocytogenes; and Candida albicans, the method including contacting the microbe with an antimicrobial composition comprising an extract from at least one plant belonging to the genus Cyclopia.

9. A method of preventing or treating a microbial infection or contamination, the method comprising applying to a substrate or surface an effective amount of an antimicrobial composition comprising an extract from at least one plant belonging to the genus Cyclopia.

10. The method according to either claim 8 or claim 9, wherein the plant is either Cyclopia subternata or Cyclopia genistoides.

11. The method according to either claim 9 or claim 10, wherein the extract is produced from the leaves and stems of the plant.

12. The method according any one of claims 8 to 10, wherein a solvent used to perform the extraction which is selected from the group including water, ethanol, and ethyl acetate.

13. The method according to any one of claims 8 to 11 , wherein the concentration of the extract is in the range of 0.5 mg/ml to 800 mg/ml.

14. The method according to claim 13, wherein the concentration of the extract is in the range of 10 mg/ml to 50 mg/ml.

15. The method according to any of one claims 8 to 13, wherein the extract is present in a ratio in the range of 2:3 to 3:2