New alternatives to cosmetics preservation

This paper describes the preservative efficacy of the well-known antimicrobial extracts of Lonicera caprifoleum and Lonicera japonica in combination with glyceryl caprylate and/or levulinic acid, p-anisic acid, and ethanol. We prepared a series of acidic (pH=5.5) aqueous and O/W formulations, i.e., tonic lotion, shampoo, shower gel, conditioning cream, anticellulite cream, cleansing milk and peeling cream, containing (0.2% w/w) Lonicera extracts, alone in the case of tonic lotion and in combination with (1% w/w) glyceryl caprylate in the other products, and we performed challenge tests according to the European Pharmacopoeia procedures and criteria. Formulations such as shampoo, shower gel, and conditioning cream fulfilled criterion A, while tonic lotion, anticellulite cream, cleansing milk, and peeling cream fulfilled criterion B, in regard to contamination from Aspergillus niger. Furthermore, we evaluated the efficacy of the antimicrobial systems in two states of use: the intact product and after three weeks of consumer use. The results showed that A. niger was also detected during use by consumers in the products that satisfied only criterion B in challenge tests. The addition of antimicrobial fragrance ingredients such as (?0.3% w/w) levulinic acid or (0.1% w/w) p-anisic acid and/or (5% w/w) ethanol afforded products that met criterion A in challenge tests and were also microbiologically safe during use. The small quantity (5% w/w) of ethanol gave an important assistance in order to boost the self-preserving system and to produce stable and safe products.

 Microbial spoilage of cosmetic formulations has always been of special concern for industry, since it can lead to product degradation or, in the case of pathogens, constitute a threat to consumer safety. Chemical preservatives are added to cosmetics, pharmaceuticals, and foods in order to protect them against microbial contamination. The growing skepticism of consumers regarding the safety of chemical preservatives in combination with the fact that long-lasting skin health is often associated with the use of natural ingredients has led the cosmetics industry to seek alternative approaches for cosmetics preservation.1,2 A recent trend in cosmetics preservation is the replacement of traditional chemical preservatives by antimicrobial agents that are not legislated as preservatives according to Annex VI of Commission Directive 1976/768/EEC3-5 but that are safe and effective as preservatives. An approach to acheive preservative-free cosmetics is the selection of natural compounds that have been characterised as safe and effective against microorganisms, in order to decrease or to eliminate the use of the traditional chemical preservatives and to formulate cosmetics with improved dermocosmetic properties, i.e., lower skin irritation and/or contact sensitisation. A number of well-known plant-derived essential oils and extracts have exhibited excellent antimicrobial properties; thus, they have been used for the effective preservation of cosmetic formulations. Among others, derivatives of Rosmarinus officinalis,7,8 Lavandula officinalis,9 Pteronia incana,8 Artemisia afra,8 Thymus vulgaris,10,11 Eucalyptus globulus,12 Laurus nobilis,12 Salvia officinalis,12 and Melaleuca alternifolia13,14 have been reported to be effective natural preservatives. Medium polar substances also belong to the class of alternative antimicrobial agents. Examples of such agents as caprylyl glycol and monoglycerides of capric acid and caprylic acid, i.e., glyceryl caprate and glyceryl caprylate, besides being moisturising agents, exert antimicrobial activities.15–17 Due to their emulsifier-like structure, with a hydrophilic and lipophilic part, they interfere with the cellular structures of microorganisms and disintegrate cell membranes. Many studies have been reported concerning the use of glyceryl caprylate as an antimicrobial substance alone or in combination with other antimicrobial compounds for cosmetics preservation.17–19 We have to keep in mind that the chemical composition of fragrances plays a key role concerning the antimicrobial activity of essential oils and the extracts obtained from natural sources.20 Various aldehydes and alcohols, i.e., aromatic and aliphatic compounds, or terpenes and organic acids, are among the most active compounds. In the past, a fragrance mixture that mainly consisted of benzyl acetate, phenethyl alcohol, and linalool had been proposed as an alternative preservative in order to reduce the amount of parabens used in cosmetic formulations.21 Today, several antimicrobial fragrance ingredients are commercially available, such as, p-anisic acid (p-methoxy-benzoic acid) and levulinic acid (4-oxo-pentanoic acid), which were found to be the main compounds in Pimpinella anisum and other herbs and in Dioscorea villosa as a by-product in the production of diosgenin from wild yam, respectively.22 Based on the above comments concerning the development of selfpreserving cosmetics, we focused our research to evaluate the preservative efficacy of the antimicrobial extracts of Lonicera caprifoleum and Lonicera japonica (Table 1) in combination with other antimicrobials such as glyceryl caprylate, p-anisic acid, levulinic acid (Table 2), and ethanol in a series of aqueous and O/W emulsions. Lonicera extracts are described as being a mixture of esters of lonicerin and p-hydroxy benzoic acid, the structures of which are very similar to those of parabens.23 Although Lonicera caprifoleum and Lonicera japonica extracts are well known for their antimicrobial properties,24–26 there are no studies in the literature regarding their incorporation as preservatives in cosmetic formulations. In order to evaluate the preservative efficacy of these multifunctional ingredients, we performed challenge tests (preservative efficacy tests, PETs) according to the standards proposed by the European Pharmacopoeia. Furthermore, we examined the microbial purity of the formulations in two different states of use (the intact product and following use) because few published papers refer to the efficacy of preservative systems contained in cosmetic products during their use by consumers.27,28

Materials and methods

Cosmetic formulations

A series of aqueous formulations, i.e., tonic lotion, shampoo, and shower gel, and O/W cosmetic formulations such as conditioning cream, anticellulite cream, cleansing milk, and peeling cream was prepared.

Tonic lotion: Water, Syringa vulgaris (lilac) extract, lactic acid, cinnamyl alcohol, hydroxycitronellal, and preservative systems I, II, III, or IV (Table 3) were used as the ingredients in the tonic lotion formulation.

Shampoo: Water, sodium cocoyl isethionate, lauryl glycoside, cocamidopropyl betaine, cocobetaine, glyceryl oleate, coco glycoside, hydrolysed milk protein, sodium phytate, Urtica dioica leaf water, Rosmarinus officinalis (rosemary) leaf water, Salix alba (white willow) leaf water, Ginkgo biloba leaf water, citric acid, fragrance, linalool, and preservative system V (Table 3) were used as the shampoo ingredients.

Shower gel: The ingredients used in the shower gel formulation were: water, sodium cocoyl isethionate, lauryl glycoside, cocamidopropyl betaine, sodium lauryl glutamate, glyceryl oleate, cocoglucoside, cocobetaine, sodium phytate, Aloe barbadensis (Aloe vera) extract, Avena sativa (oat) leaf extract, Calendula officinalis leaf water, Arnica montana leaf water, Lavandula angustifolia (lavender) leaf water, hydrolysed milk protein, parfum (fragrance), citric acid, D-limonene, and preservative system V (Table 3).

Conditioning cream: The conditioning cream ingredients were: water, hydroxypropyl starch phosphate, cetyl alcohol, dioleyloylethyl hydroxyethylammonium methosulfate, sucrose laurate, cetearyl alcohol, Macadamia ternifolia seed oil, glycerin, polyglyceryl-10 laurate, meadowfoam (Limnantes alba) seed oil, fragrance, stearyl stearate, glycine soja, phospholipids, soy sterol, sodium phytate, ethanol, lauryl glycoside, tocopheryl acetate, Urtica dioica (nettle) leaf water, Rosmarinus officinalis (rosemary) leaf water, Gingko biloba leaf water, Salix alba (white willow) leaf water, hydrolysed milk protein, tocopherol, and preservative system V (Table 3).

Anticellulite cream: The ingredients used in the anticellulite cream formulation were: water, sodium stearoyl lactylate, caffeine, glycerin, tricaprylin, dicaprylyl carbonate, isopropyl myristate, polyglyceryl- 3 stearate, dicaprylyl ether, cetyl alcohol, glyceryl stearate, panthenol, behenyl alcohol, glyceryl stearate, lecithin, glycine soja (soybean) sterols, Lactobacillus/ Trifolium pratense (clover) flower ferment extract, Lactobacillus/Theobroma cacao (cocoa) ferment extract, Lactobacillus/ Camellia sinensis leaf ferment extract, Vitis vinifera (grape) seed oil, Prunus armeniaca (apricot) kernel oil, sodium phytate, xanthan gum, escin, tocopherol, citric acid, Olea europaea (olive) fruit extract, parfum (fragrance), linalool, benzyl benzoate, benzyl salicylate, farnesol, geraniol, eugenol, and preservative system V, VI, or VII (Table 3).

Cleansing milk: The cleansing milk ingredients were: water, isopropyl myristate, glyceryl stearate, polyglyceryl 3-stearate, myristyl myristate, glycerin, sodium stearoyl lactylate, tricaprylin, caprylic/capric triglyceride, Calendula officinalis oil, cetearyl alcohol, bisabolol, Prunus armeniaca (apricot) kernel oil, tocopherol, xanthan gum, sodium phytate, Aloe barbadensis extract, sodium hydroxide, citric acid, Chamomilla recutita (Matricaria) extract, and preservative system V, VI, VII, or VIII (Table 3).

Peeling cream: The ingredients used in the peeling cream formulation were: water, glycerin, isopropyl myristate, Prunus dulcis amygdalus (almond) shell granules, glyceryl stearate, myristyl myristate, polyglyceryl 3- stearate, tricaprylin, stearic acid, sodium stearoyl lactylate, cetyl alcohol, cetearyl alcohol, caprylic/capric triglyceride, Prunus dulcis amygdalus (almond) seed oil, tocopherol, xanthan gum, sodium stearate, Simmondsia chinensis (jojoba) seed oil, sodium phytate, sodium hydroxide, Chamomilla recutita (Matricaria) extract, citric acid, parfum (fragrance), limonene, and preservative system V, VI, or VII (Table 3).

Essential oils and multifunctional ingredients with antimicrobial activity

• Planteservative WSr (Campo Cosmetics S Pte. Ltd., Singapore) = Lonicera caprifoleum flower extract and Lonicera japonica flower extract, water.
• Dermosoft GMCY (Dr Straetmans Chemische Produkte GmbH, Hamburg, Germany) = glyceryl caprylate.
• Dermosoft 688 (Dr Straetmans Chemische Produkte GmbH, Hamburg, Germany) = p-anisic acid.
• Dermosoft 1388 (Dr Straetmans Chemische Produkte GmbH, Hamburg, Germany) = levulinic acid (10%), sodium hydroxide, glycerin, water.
• Ethanol (not denaturated).

Organisms and inoculum preparation

Organisms: Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Escherichia coli, Aspergillus niger ATCC 16404, and Candida albicans ATCC 10231 were used.

 Inoculum preparation:

For bacteria and C. albicans inoculum, the cells (108) were harvested into 0.1% peptone water by gentle agitation and adjusted to yield suspensions of approximately 106 cfu/mL. The count of A. niger (approx. 105) was achieved after the 1% (w/w) dilution of the initial suspension (107). The peptone water used for harvesting A. niger contained 0.05% v/v of Tween 80 (Sigma-Aldrich).

Microbial challenge tests (preservative efficacy tests, PETs) according to the European Pharmacopoeia (E. Ph.)

Preliminary studies were performed in order to assure the ability of the unpreserved formulations to support the viability and/or microbial growth and also the effectiveness of the neutralising medium for the inoculum recovery. The microbial challenge test was performed according to the standards proposed by the European Pharmacopoeia (E. Ph., 1996) concerning topical preparations. The formulations (samples of 20 g) were placed in sterile containers and separately inoculated with bacterial and fungal suspensions to reach microbial levels of not less than 106 cfu/g for bacteria and 105 cfu/g for fungi. The test samples were mixed, diluted in Letheen broth, and assayed at 0, 2, 7, 14, 21, and 28 days. The assays were performed on 1 g or 1 mL of test sample and plated in triptic soy agar and Sabouraud dextroze agar for bacteria and fungi, respectively. Plates were incubated at 35°C for bacteria and at 25°C for fungi. After a five-day incubation, colonies of bacteria and fungi were counted and (cfu/g) calculated. The experiments were performed in triplicate. Products are judged adequately preserved when bacteria are reduced by more than 99% (2 log) after two days and more than 99.9% (3 log) after seven days; yeasts and moulds should be reduced by more than 99% (2 log for criterion A and 1 log for criterion B) after 14 days.

Microbiological quality in two states of use: intact product and following three weeks of use

The collected samples of cosmetic products were analysed for total aerobic plate count (S. aureus, P. aeruginosa, E. coli, A. niger, and C. albicans) in two different states of use, the intact product and after three weeks of use. One gram or 1 mL of test sample was serially diluted in Letheen broth and plated in triptic soy agar and Sabouraud dextroze agar for bacteria and fungi, respectively. Plates were incubated at 35°C for bacteria and at 25°C for fungi. After a five-day incubation, colonies of bacteria and fungi were counted and (cfu/g) calculated. The experiments were performed in triplicate. In some cases A. niger was identified as black colonies with the characteristic morphology of actinomycetes.

Results

Microbiological quality of the test product The results regarding the microbiological safety of the formulations tested (challenge test according E. Ph., intact products, and following three weeks of use) are summarised in Tables IV, V, VIa–e, VII and VIII and in Figures 1 and 2.

Staphylococcus aureus: Systems I–IV preserved effectively the high-watercontaining tonic lotion against this strain (Table 4). System V could not be used in the case of the tonic lotion due to solubility problems, but it was active in the cases of shampoo and shower gel and all the emulsified formulations tested (Fig. 1, Tables IV and VIa). Preservative systems VI and VII showed excellent activity in all the O/W formulations, i.e., the anticellulite cream, cleansing milk, and peeling cream. System VIII with the reduced percentage (0.1% w/w) of levulinic acid also proved to be effective. In all above cases, criterion A of the E. Ph. were fulfilled. Furthermore, no contamination from this strain was found, either in the intact products or following consumer use (Tables 5, 7 and 8).

Pseudomonas aeruginosa: This Gramnegative microorganism was susceptible to preservative systems I–IV in the case of the tonic lotion (Table 4). System V effectively protected the shampoo, shower gel, and O/W preparations. (Fig. 1, Tables 4 and 6b). System VI also preserved equally to system V all the O/W formulations tested, i.e., the anticellulite cream, cleansing milk, and peeling cream. As with the previous microorganism, levulinic acid (0.3% w/w, system VII, or 0.1% w/w, system VIII), was effective. In all of the above cases, criterion A of the E. Ph. was met. Additionally, no contamination was found either in the intact products or following three weeks of use (Tables V, VII, and VIII).

Escherichia coli: The population of this test microorganism seemed to be effectively controlled by systems I–IV in the case of the tonic lotion, since criterion A of E. Ph. was satisfied (Table 4). System V was also active in the shampoo and shower gel and the emulsified formulations (Fig. 1, Tables 4 and 6c). Preservative systems VI and VII revealed excellent activity against this microorganism in the O/W formulations tested, i.e., the anticellulite cream, cleansing milk, and peeling cream. System VIII, with the reduced percentage of levulinic acid, was sufficient as well. The strain was not detected in the intact product (Tables V and VII). Furthermore, the strain was not recovered during the inuse consumer use test (Tables 5 and 8).

Aspergillus niger: System I preserved marginally the tonic lotion against this mould in the challenge test, since only criterion B of E. Ph. was achieved (Fig. 2 and Table 4). Although no contamination of the mould was detected in the intact product (Table 7), recovery was observed during the in-use study (Table 8). Addition of (0.1% w/w) p-anisic acid (system II) or (0.3% w/w) levulinic acid (system III) or (5% w/w) ethanol (system IV) to Lonicera extracts (system I) enhanced the efficacy, and the resulting products were microbiologically safe either in the challenge test (Fig. 2 and Table 4) or during the consumer-use study (Tables 7 and 8). System V preserved efficiently the shampoo and shower gel (Fig. 1, Tables IV and VId). O/W formulations, besides the conditioning cream, were not completely protected against A. niger with system V, since in the cases of anticellulite cream, cleansing milk, and peeling cream only criterion B of E. Ph. was fulfilled (Fig. 1, Tables IV and VId). The relative activity exerted previously by system V against the three bacterial strains did not seem to affect this fungus in the emulsified formulations tested. A population of about 103 cfu/g was counted at the end of the challenge test (Fig. 1d), whereas recovery was observed following three weeks of use (Table 8). The addition of p-anisic acid (system VI) or levulinic acid (systems VII and VIII) inhibited the growth of the mould in the O/W formulations, i.e., the anticellulite cream, cleansing milk, and peeling cream, and criterion A was achieved in the challenge test. No contamination of the mould was detected either in the intact product or following use (Tables 5, 7, and 8), when systems VI, VII, or VIII were utilised.

Candida albicans: System I fulfilled marginally criterion B in the case of the tonic lotion in the challenge test (Table 4). Systems II–IV were effective against yeast in the tonic lotion during the challenge test (Table 4) and in the in-use study (Tables 5, 7, and 8). Systems V (Fig. 1, Tables 4 and 6e) and VI (Table 4) were active against yeast in the O/W formulations tested. Systems VII and VIII with (0.3% w/w) levulinic acid and (0.1% w/w) p-anisic acid, respectively, preserved sufficiently the emulsified products and criterion A was fulfilled. No contamination from the mould was detected either in the intact product or during the in-use consumer study (Tables 5, 7, and 8).

Physicochemical stability

The results concerning physicochemical stability are summarised in Table 4. Lonicera extracts alone (system I) was used only in the case of the tonic lotion and did not cause stability problems. The addition of (0.1% w/w) p-anisic acid or (0.3% w/w) levulinic acid to system I (i.e, systems II and III), caused the precipitation of solids after a few days. System V afforded stable O/W emulsions, the shampoo and the shower gel. However, it could not be used in the case of the tonic lotion, where the water content was high, due to the solubility problems of glyceryl caprylate. Furthermore, the enrichment of system V with (0.1% w/w) anisic acid (system VI) or (0.3% w/w) levulinic acid (system VII) led to the separation of the phases in the cases of the cleansing milk and the anticellulite cream after 20 days. In contrast, addition of (0.1% w/w) levulinic acid (system VIII) did not influence the stability of the cleansing milk.

Water activity

Water activity (aw) or equilibrium relative humidity quantifies the active part of the moisture content or “free water” as opposed to the total moisture content, which also includes “bound water.” It indicates the amount of water in the total water content that is available to microorganisms. Each species of microorganism has its own minimum aw value below which growth is no longer possible.6 The results of water activity measurements of the tested formulations are presented in Table 4.

Discussion

All the tested antimicrobial systems (I–VIII) have exerted excellent activity against Gram-positive and Gram-negative bacteria in the acidic (pH=5.5) environment used. They protected efficiently the emulsified and aqueous formulations against Grampositive and Gram-negative bacteria in challenge tests (criterion A of E. Ph.) and in in-use study (intact products and following three weeks of use). Antimicrobials I–VIII proved to be effective against Gram-positive bacteria, although 0.86 is the lowest aw value permitting S. aureus growth. Acidic pH conditions may contribute to the increase in the minimum aw value for this microorganism6 and therefore improve the performance of alternative systems used. Of course, manipulation of aw is only part of the preservative system. The activity against Gram-negative bacteria could be partially attributed to the relatively low water activity values of the products (0.865–0.932) (Table 6), since water activity values lower than 0.95 prevent the growth of Gramnegative microorganisms.6 We note that these microorganisms are known to be very persistent and often are recovered in the in-use state, probably from the hands of the consumers,27 even in products containing effective traditional preservatives such as parabens and phenoxyethanol.28 On the other hand, fungi were less susceptible to Lonicera extracts (system I) and Lonicera extracts/glyceryl caprylate (system V). Although, system I in the case of the tonic lotion showed moderate activity against A. niger and C. albicans in challenge tests (criterion B of E. Ph.), significant levels of mould were recovered following use. The moderate efficacy of system I in the preservation of the tonic lotion could be ascribed to the inability of Lonicera extracts (0.2% w/w) to inhibit mould in this formulation. System V protected the aqueous shampoo and shower gel against A. niger, where criterion A of E. Ph. was fulfilled. Probably the antimicrobial potencies of Rosmarinus officinalis leaf water in the shampoo and Lavender angustifolia leaf water in the shower gel enhanced the antifungal activity of system V. System V satisfied marginally criterion B of E. Ph against A. niger in all the emulsified formulations except the conditioning cream. The greater ability of system V in this cosmetic form could be ascribed to cationic agents, which might reinforce the antimicrobial activity.28,29 Furthermore, system V was unable to preserve the anticellulite cream, cleansing milk, and peeling cream, since A. niger was detected after three weeks of consumer use. Another factor that might enhance contamination risk in the case of the peeling cream is the jar-container, which allows the entry of microorganisms into the product. The lack of efficacy in system V against A. niger in some emulsified formulations is in accordance with the findings reported previously that the preservative performance of glyceryl caprylate against moulds depends on the formulation.18,19 Regarding the fragrance ingredients, although the addition of (0.1% w/w) anisic acid (systems II and VI) or (0.3% w/w) levulinic acid (systems III and VII) significantly improved antifungal activity, in some cases it caused stability problems. The reduction of the concentration of levulinic acid to 0.1% w/w resulted in microbiologically and physicochemically stable products.

Conclusion

The results demonstrate that natural origin ingredients such as Lonicera extracts seem to be promising as antimicrobial substances for producing self-preserving cosmetic products. The addition of multifunctional ingredients such as glyceryl caprylate, levulinic acid or p-anisic acid and/or ethanol was beneficial in the majority of the products. Ethanol at low concentration (i.e., 5% w/w) may contribute to the performance of the antimicrobial. An interesting observation is that the products that fulfilled criterion B in the challenge tests proved to be inadequately preserved after of three weeks of consumer use. We note that this is not surprising since criterion B is more lenient than criterion A. We suggest that E. Ph. should be changed to recognise only criterion A for adequately preserved products in multipleuse containers. Furthermore, challenge tests should be performed not only during the preparation of cosmetic products, but should also be used to evaluate the protection efficacy of the preservative systems following periods of use.

• This article was originally published by Journal of Cosmetic Science in 2010.

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