Efficiency of Calamintha officinalis essential oil as preservative in two topical product types

Authors


A. Nostro, Dipartimento Farmaco-Biologico, Sezione Microbiologia, Facoltà di Farmacia, Università di Messina, Villaggio Annunziata, 98168, Italy (e-mail: atnostro@pharma.unime.it).

Abstract

Aims:  To verify the efficiency of Calamintha officinalis essential oil as natural preservative in two current formulations.

Methods and Results:  The 1·0 and 2·0% (v/v) C. officinalis essential oil was assayed for its preservative activity in two product types (cream and shampoo). The microbial challenge test was performed following the standards proposed by the European Pharmacopoeia Commission (E.P.) concerning topical preparations using standard micro-organisms and in addition wild strains, either in single or mixed cultures were used. The results clearly demonstrated that the C. officinalis essential oil at 2·0% concentration reduced the microbial inoculum satisfying the criterion A of the E.P. in the cream formulation and the criterion B in the shampoo formulation. Standard and wild strains showed a behaviour similar, both in cream and in shampoo formulation, with no significant difference (gerarchic variance, P > 0·05).

Conclusion: C. officinalis essential oil confirmed its preservative properties but at higher concentration than that shown in previous studies on cetomacrogol cream.

Significance and Impact of the Study:  The nature of the formulation in which an essential oil is incorporated as preservative could have considerable effect on its efficacy.

Introduction

The microbiological safety of a pharmaceutical and cosmetic product has always been of special interest for industries as a microbial contamination can lead to product degradation or, in the case of pathogens, constitute a risk for the health of the consumer and potentially spread infection (Bloomfield 1990; Wong et al. 2000). Preservatives are included in pharmaceutical and cosmetic formulations to protect the product from microbial insults occurring from raw materials, manufacture and consumer use. The cosmetics industry relies on microbial challenge testing as to evaluate how well a product withstands microbial insults and, therefore, it is important that this testing to be a valid predictor should include, in addition to standard micro-organisms, also wild strains which are generally believed to be more aggressive than laboratory strains and are more representative of potential contaminants that a product might encounter during use (Zani et al. 1997).

A recent trend in cosmetic preservation is to avoid the use of chemical agents, leaving scientists in the search for natural antimicrobial alternatives (Johnson et al. 2000; Seo et al. 2002). Plant-derived essential oils possess potential as natural agents for cosmetic preservation due to their versatile content of antimicrobial compounds (Kabara 1984; Manou et al. 1998). Lately, the essential oil of Calamintha officinalis Moench (Lamiaceae), a plant commonly found especially in dry places and used for its diaphoretic, expectorant, and flavouring properties (Grieve 1981) has been reported for its preservative properties in culture medium and in cetomacrogol cream (Nostro et al. 2002). Carvone, its major constituent, is widely used as a cosmetics flavouring agent and has been patented for its use as a skin penetration enhancer (Leonard et al. 1989; Fuchs et al. 1997; Gao and Singh 1997). In this paper, we performed a more realistic evaluation of C. officinalis essential oil preservative properties using two current formulations, which have also been challenged with wild strains, in single or mixed cultures, besides standard micro-organisms. The formulations were developed on the basis of new demands of the market towards the product formulation through a ‘cold process’, i.e. with less expenditure of energy, shorter preparation time and consequently reduction of exposure to high temperatures.

Materials and methods

Preparation of test formulation

The Calamintha officinalis Moench essential oil was hydrodistilled as previously reported (Nostro et al. 2002). Two product types, cream and shampoo, were evaluated.

The cream composed of water, liquid paraffin 15%, glycerin 2·0%, sodium polyacrylate 0·7%, EDTA 0·09%. Four preservative conditions were performed: cream preserved with essential oil 2·0%, v/w; cream preserved with essential oil 1·0%, v/w; cream preserved with with a fluid active combination of phenonip® (Nipa Laboratories, Hengoed, UK) (phenoxyethanol, methylparaben, ethyl paraben, propyl parben and diethyl parben) 0·5% (positive control); cream without any preservative (negative control).

The shampoo was composed of water, magnesium laureth sulphate 25%, disodium laureth sulphosuccinate 5·0%, lactoglobulin 5·0%, EDTA 0·09%. Three shampoos were prepared with different preservative systems: shampoo preserved with essential oil 2·0%, v/v; shampoo preserved with a fluid active combination of phenonip® 0·5% (positive control); shampoo without any preservative (negative control).

The ingredients used in the manufacture of these products were standard cosmetic grade ingredients.

Organisms and inocula preparation

Organisms.

The micro-organisms used in this study include strains from ATCC culture collections, strains isolated from spoilt preparations and clinical isolates. They were the following: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 9027), Ps. aeruginosa (cream isolate), Burkholderia cepacia (ATCC 25416), Staphylococcus aureus (ATCC 6538P) Staph. aureus (methicillin-resistant Staph. aureus MRSA, clinical isolate,), Staph. epidermidis (ATCC 12228), Candida albicans (ATCC 10231) C. albicans (clinical isolate) and Aspergillus niger (ATCC 16404).

Inocula preparation.

Bacteria were cultured on Tryptone Soya Agar (TSA; Oxoid, Milan, Italy) for 24 h at 37°C. C. albicans and A. niger were grown on Sabouraud Dextrose Agar (SDA; Oxoid) at 25°C for respectively 48 h and 5 days. For microbial inocula, the cells were harvested into 0·1% peptone water by gentle agitation and adjusted to yield suspensions of approx. 108 CFU ml−1 or 108 spores ml−1, using turbidimetry absorbance correlated to an aerobic plate count. The peptone water used for harvesting A. niger contained 0·05% (v/v) of Tween-80 (Sigma-Aldrich, Milan, Italy).

Microbial challenge test

Preliminary studies were performed in order to assure the ability of the unpreserved formulations to support the viability and/or the microbial growth and also the effectiveness of the neutralizing medium for the inoculum recovery.

The microbial challenge test was performed following the standards proposed by the European Pharmacopoeia Commission (1996) concerning topical preparations and considering in addition to standard micro-organisms also wild strains either in single or mixed cultures.

Single inoculum.

The formulations (samples of 20 g) were placed in sterile containers and separately inoculated with bacterial and fungal suspensions to give a final level of approx. 106 CFU g−1. After a contact time of 0, 2·0, 7·0, 14, and 28 days, at 20–25°C the samples (1·0 g) were removed and placed into 9·0 ml of neutralizing medium Leethen broth (Difco). Cell viability was determined by the plate count method in TSA or SDA and CFU were counted after a 5-day incubation at 37 and 25°C for bacteria and mycetes respectively. The results were confirmed in three different experiments.

Mixed cultures.

Equal parts of the Staph. aureus and Staph. epidermidis suspensions were placed in a sterile container to make inoculum of mixed cultures of Gram-positive bacteria. Equal parts of the Ps. aeruginosa and B. cepacia suspensions were placed in a sterile container to make inoculum of mixed cultures of Gram-negative bacteria. Equal parts of the C. albicans and A. niger suspensions were placed in a sterile container to make inoculum of mixed cultures of mycetes.

The preparations (samples of 20 g) were placed in sterile containers and separately inoculated with each mixed culture to give a final level of approx. 106 CFU g−1. After a contact time of 0, 2·0, 7·0, 14, and 28 days, the samples (1·0 g) were removed and counted according to the method described above. The results were confirmed in three different experiments.

The viability of the inoculated cells and their ability to grow were evaluated by a growth control consisting of cream without essential oil.

Statistical analysis

The data were subjected to analysis of gerarchic anova test to determine the significant difference between the standard and wild strains as well as between the single and mixed cultures. The results were considered statistically significant when P < 0·05.

Results

The microbial challenge test results are shown in Tables 1–4. The bacterial challenge test performed in cream formulation preserved with C. officinalis at 2·0% concentration satisfied the E.P. criteria A (i.e. reduction of the bacterial inoculum by a factor of 103 within 7 days of challenge with no increase up to the 28th day) with no statistical difference between standard and wild strains (P > 0·05). In particular, C. officinalis at 2·0% concentration reduced the bacterial inoculum by a factor of 103 for Gram-positive and even of almost 104 for Gram-negative within 7 days. On the other hand, C. officinalis at 1·0% concentration only satisfied the E.P. criteria B (reduction by a factor of 103 within 14 days of challenge with no increase up to the 28th day) being ineffective as regards the criterion A for Staph. aureus standard and wild strain (ATCC 6538P and MRSA) whose growth decreased gradually during the incubation time. The control composed of cream traditionally preserved with phenonip satisfied the E.P. criterion A whereas the unpreserved base cream failed the test.

Table 1.  Effectiveness of C. officinalis essential oil (EO) in cream formulation: challenge test with single cultures
StrainsCreamTime (day) (log CFU g−1)
027142128
Staph. aureus ATCC 6538PUnpreserved5·55·55·155·05·05·0
EO 1·0% (v/w) preserved5·53·73·22·62·01·0
EO 2·0% (v/w) preserved5·53·02·02·01·01·0
Phenonip® preserved5·52·02·01·01·01·0
Staph. aureus MRSA, clinical isolateUnpreserved6·05·65·65·05·05·0
EO 1·0% (v/w) preserved6·05·03·52·82·52·0
EO 2·0% (v/w) preserved6·03·62·92·11·01·0
Phenonip® preserved6·02·02·01·01·01·0
Staph. epidermidis ATCC 12228Unpreserved6·06·05·65·55·34·0
EO 1·0% (v/w) preserved6·03·63·82·42·01·0
EO 2·0% (v/w) preserved6·03·02·21·01·01·0
Phenonip® preserved6·02·01·01·01·01·0
E. coli ATCC 25922Unpreserved6·56·56·46·45·85·4
EO 1·0% (v/w) preserved6·53·82·52·21·71·0
EO 2·0% (v/w) preserved6·53·21·21·01·01·0
Phenonip preserved6·52·01·01·01·01·0
Ps. aeruginosa ATCC 9027Unpreserved6·26·26·05·85·05·0
EO 1·0% (v/w) preserved6·23·32·21·01·01·0
EO 2·0% (v/w) preserved6·22·51·51·01·01·0
Phenonip® preserved6·22·01·01·01·01·0
Ps. aeruginosa cream isolateUnpreserved6·46·46·36·35·85·4
EO 1·0% (v/w) preserved6·43·42·41·81·01·0
EO 2·0% (v/w) preserved6·42·81·51·01·01·0
Phenonip® preserved6·41·01·01·01·01·0
B. cepacia ATCC 25416Unpreserved7·16·56·56·46·26·2
EO 1·0% (v/w) preserved7·13·53·02·81·11·1
EO 2·0% (v/w) preserved7·13·01·61·11·11·1
Phenonip® preserved7·12·71·11·11·11·1
C. albicans ATCC 10231Unpreserved6·05·55·56·06·06·0
EO 1·0% (v/w) preserved6·03·32·01·71·51·0
EO 2·0% (v/w) preserved6·02·01·01·01·01·0
Phenonip® preserved6·02·01·01·01·01·0
C. albicans clinical isolateUnpreserved6·06·06·06·26·26·2
EO 1·0% (v/w) preserved6·04·53·83·02·82·0
EO 2·0% (v/w) preserved6·03·73·22·82·21·8
Phenonip® preserved6·01·01·01·01·01·0
A. niger ATCC 16404Unpreserved6·54·84·64·64·64·6
EO 1·0% (v/w) preserved6·54·84·24·04·04·0
EO 2·0% (v/w) preserved6·54·64·04·04·04·0
Phenonip® preserved6·51·01·01·01·01·0
Table 2.  Effectiveness of C. officinalis essential oil (EO) in cream formulation: challenge test with mixed cultures
StrainsCreamTime (day) (log CFU g−1)
027142128
Gram-positive bacteria
 Staph. aureus ATCC 6538P + Staph. epidermidis ATCC 12228Unpreserved5·35·35·25·25·05·0
EO 1·0% (v/w) preserved5·33·63·01·81·41·0
EO 2·0% (v/w) preserved5·33·02·01·01·01·0
Phenonip® preserved5·31·91·01·01·01·0
Gram-negative bacteria
 Ps. aeruginosa ATCC 9027 + B. cepacia ATCC 25416Unpreserved6·46·46·46·46·46·4
EO 1·0% (v/w) preserved6·43·52·42·02·01·0
EO 2·0% (v/w) preserved6·42·82·01·01·01·0
Phenonip® preserved6·42·81·01·01·01·0
Mycetes
 C. albicans ATCC 10231 + A. niger ATCC 16404Unpreserved6·46·35·45·45·45·4
EO 1·0% (v/w) preserved6·44·84·54·14·03·8
EO 2·0% (v/w) preserved6·44·14·13·94·03·5
Phenonip® preserved6·42·01·01·01·01·0
Table 3.  Effectiveness of C. officinalis essential oil (EO) in shampoo formulation: challenge test with single cultures
StrainsShampooTime (day) (log CFU g−1)
027142128
Staph. aureus ATCC 6538PUnpreserved5·45·34·84·23·02·4
EO 2·0% (v/w) preserved5·44·62·82·01·51·0
Phenonip® preserved5·44·22·21·01·01·0
Staph. aureus MRSA, clinical isolateUnpreserved6·05·54·93·83·52·5
EO 2·0% (v/w) preserved6·04·54·03·02·51·8
Phenonip® preserved6·04·02·21·01·01·0
Staph. epidermidis ATCC 12228Unpreserved5·75·15·04·53·22·5
EO 2·0% (v/w) preserved5·74·02·52·01·31·0
Phenonip® preserved5·73·02·01·01·01·0
E. coli ATCC 25922Unpreserved5·55·85·96·06·27·0
EO 2·0% (v/w) preserved5·52·72·32·01·01·0
Phenonip® preserved5·52·72·01·01·01·0
Ps. aeruginosa ATCC 9027Unpreserved6·05·05·56·06·87·0
EO 2·0% (v/w) preserved6·02·22·21·01·01·0
Phenonip® preserved6·02·22·01·01·01·0
Ps. aeruginosa cream isolateUnpreserved5·46·06·66·97·07·2
EO 2·0% (v/w) preserved5·42·12·01·01·01·0
Phenonip® preserved5·42·22·01·01·01·0
B. cepacia ATCC 25416Unpreserved5·66·57·07·27·27·5
EO 2·0% (v/w) preserved5·62·22·01·01·01·0
Phenonip® preserved5·62·01·01·01·01·0
C. albicans ATCC 10231Unpreserved5·14·63·53·32·82·0
EO 2·0% (v/w) preserved5·13·12·82·11·11·1
Phenonip® preserved5·12·11·81·11·11·1
C. albicans clinical isolateUnpreserved5·64·95·04·03·22·5
EO 2·0% (v/w) preserved5·63·53·32·82·01·0
Phenonip® preserved5·63·02·01·01·01·0
A. niger ATCC 16404Unpreserved5·64·54·64·24·03·0
EO 2·0% (v/w) preserved5·63·43·03·02·02·0
Phenonip® preserved5·62·02·01·01·01·0
Table 4.  Effectiveness of C. officinalis essential oil (EO) in shampoo formulation: challenge test with mixed cultures
StrainsShampooTime (day) (log CFU g−1)
027142128
  1. ‘–’, Not determined.

Gram-positive bacteria
 Staph. aureus ATCC 6538P + Staph. epidermidis ATCC 12228Unpreserved6·04·74·03·02·52·4
EO 2·0% preserved6·04·62·01·51·01·0
Phenonip® preserved6·04·01·01·01·01·0
Gram-negative bacteria
 Ps. aeruginosa ATCC 9027 + B. cepacia ATCC 25416Unpreserved6·05·55·86·06·57·0
EO 2·0% preserved6·03·02·01·01·01·0
Phenonip® preserved6·02·81·81·01·01·0
Mycetes
 C. albicans ATCC + 10231 A. niger ATCC 16404Unpreserved
EO 2·0% preserved
Phenonip® preserved

The examination of E.P. criteria A for mycetes (i.e. reduction by a factor of 102 within 14 days of challenge with no increase up to the 28th day) revealed that the log reduction in the number of viable micro-organisms was acceptable for both C. officinalis essential oil 1·0 and 2·0% concentration. In spite of this, except for C. albicans standard strain (ATCC 10231) that showed a good susceptibility, C. albicans wild strain and A. niger standard strain (ATCC 16404), showed a certain resistance which remained inaltered through the whole process whatever the concentration.

The challenge testing performed in shampoo formulation revealed that the shampoo preserved with C. officinalis at 2·0% concentration showed a reduction of the inoculum satisfying the E.P. criterion B for all micro-organisms with no statistical difference between standard and wild strains (P > 0·05). The criterion A were not satisfied for Staph. aureus standard and wild strains (ATCC 6538P and MRSA). The positive control constituted by shampoo traditionally preserved with phenonip® met the E.P. criterion A whereas the negative control (unpreserved shampoo) proved a good substrate for the Gram-negative bacteria with bacterial numbers increasing over 28 days and a biocide for Gram-positive bacteria and mycetes that were progressively reduced. This antimicrobial effect, however, was slower and less severe than the one observed in shampoo preserved with essential oil.

Mixed culture showed a behaviour similar to single cultures both in cream and in shampoo formulation. The statistical analysis (gerarchic variances) showed no significant differences (P > 0·05).

Discussion

The present work investigated the preservative efficiency of C. officinalis essential oil in two current formulations. It was demonstrated that this essential oil confirmed its preservative properties, with no significant differences (P > 0·05) between standard and wild strains either in single or mixed cultures, but the nature of formulation in which it was incorporated had considerable effect on its efficacy. The cream formulation containing liquid paraffin as major component and essential oil at 2·0% concentration was well preserved (in accordance with E.P. criterion A) and achieved the inhibition of all micro-organisms challenged. This higher concentration (2·0%) of C. officinalis compared with concentration of 1·0%, which had previously been used as preservatives in cetomacrogol cream (Nostro et al. 2002), could be explained by the lipophilic affinity of the essential oil for liquid paraffin. When added to a formulation, the preservative distributes itself into the oil and aqueous phases and its antimicrobial activity is largely attributed to its free concentration in the last phase. Although essential oils are often thought highly lipophilic compounds insoluble in the aqueous phases, in effect, in many cases they have a relative hydrophilicity given by the presence of constituents with polar functional groups. Carvone, the major component of C. officinalis essential oil (Nostro et al. 2002) has a water solubility of 916 ± 20 ppm and belongs to group I of compounds able to form a hydrogen bond (Griffin et al. 1999). The affinity of the essential oil for liquid paraffin probably reduced the essential oil bioavailability in aqueous phase, so that, a 1·0% concentration was unable to reduce Gram-positive bacteria, in particular of Staph. aureus cells (ATCC 6538P and MRSA clinical isolate). This observation was corroborated by studies of Orafidiya et al. (2001) who demonstrated that the antimicrobial activity of Ocimum gratissimum essential oil was influenced by the presence of liquid paraffin. Considering that, C. officinalis at 1·0% concentration showed unsatisfactory activity and that the microflora of the skin or consumer's hands are assumed to be a major source of contamination during skin cosmetics application, it would be advisable to utilize the essential oil at 2·0% concentration to avoid bacterial recovery and/or possible infection spreading. Besides, it is possible to recover high levels of microbial contamination by Bacillus, Micrococcus and Staphylococcus species in preserved cosmetic products, without visible biodeterioration (Flores et al. 1997).

In contrast, the antimicrobial activity of the C. officinalis essential oil was more effective against the Gram-negative bacteria, that may have been due to the presence of EDTA that acted as synergic agent. This is very important as the Gram-negative organisms are known to be very persistent and predominant in case of cosmetic spoilage (Anelich and Korsten 1996), as they largely multiply in creams and lotions also in the presence of preservatives (Malcom 1976). Thus, it is not surprising that in our unpreserved formulations there was a major persistence of Gram-negative rather than Gram-positive bacteria and even a growth in shampoo formulation.

The results of the challenge test in shampoo formulation showed that, although it had an intrinsic antimicrobial activity against the Gram-positive bacteria and mycetes, due to the presence of surfactants (Brannan et al. 1987; Orafidiya et al. 2001), the 2·0% concentration of C. officinalis essential oil was insufficient to reduce the Gram-positive inoculum, as required by the P.E. criterion A, and to determine a rapid reduction of mycetes. On the contrary, as for Gram-negative bacteria, the unpreserved shampoo was a good growth substrate whereas the essential oil-preserved shampoo showed a satisfactory preservative efficacy. Here too, interfering factors could have influenced, alone or in concert, on the oil effectiveness and caused different effects among the different organisms. Surfactants are organic compounds composed of both hydrophobic and hydrophilic parts and normally found in detergents and shampoos. They reduce surface tension, thereby diminishing interfacial forces and leading to the formation of a micellar structure. The affinity of the essential oil for the surfactant micelle could have lowered the oil bioavailability and then partially neutralized its antimicrobial activity as well as liquid paraffin in the cream formulation. On the other hand, it is known that the surfactants influence negatively the antimicrobial activity of some essential oils (Remmal et al. 1993; Orafidiya et al. 2001). In this formulation, not only the surfactants but also the presence of lactoglobulin (organic matter) may have interfered with antibacterial activity of essential oil. The protein–cell or protein–oil interactions could prevent the cell exposure to the oil or render the essential oil in a form less readily adsorbed by different micro-organisms. Some studies have reported that both organic matter and surfactants compromised the antimicrobial activity of tea tree oil (Hammer et al. 1999) as well as the bovine serum albumin neutralized the antimicrobial activity of thymol with interaction between oil and proteins (Juven et al. 1994).

Nevertheless the reduced activity of the essential oil at 2·0% concentration in shampoo formulation, it is important to point out that: first, the shampoo showed a good preservative efficacy against the very persistent Gram-negative bacteria or typical contaminants of the water, that might more easily contaminate the shampoo rather than the cream; secondly, the E.P. recommends satisfaction of criterion A, but in justified cases imposes satisfaction of criterion B. It is true, indeed, that a higher concentration of essential oil than 2·0% might resolve the problem but this might have a high impact on the organoleptic properties of formulation and thus, it should be of interest to verify the activity of C. officinalis in combination with other antimicrobial agents.

In spite of scepticism that has so far strongly influenced research as regards the use of natural substances as alternatives to many existing preservatives, the manufacture of cosmetics based on natural ingredients is becoming increasingly common. The natural compounds with their anti-inflammatory, antioxidant and antimicrobial activity could potentiate the dermo-cosmetic properties of products of skin or hair care in which they are commonly included as fragrances, soothing and cooling agents.

Ancillary