Bacteriostatic esters of 5-oxoproline

The amino acid pyroglutamic acid (PCA) (also known as 5-oxoproline) is widespread in plants and animals. It is involved in several physiological processes, such as energy production and cell building.

Like all substances of biological interest, it normally occurs as the levorotatory enantiomer, but the dextro- form and racemic mixtures are also known. Its chemical origin is attributed to the cyclization reaction of glutamic acid, a reaction from which all the possible steric isomers originate. They are characterized by the stability of their free energy state.

PCA is an intermediate of glutation synthesis through the gamma glutamic cycle. The involvement of PCA in glucidic catabolysis processes for the production of energy derives from its connection to glutation. Also known are specific interferences of the molecule on the permeability of cerebral membranes with positive consequences on learning and memorization processes.

Biological activity

PCA belongs to the so-called ‘nootropics’ family of molecules. The term is derived from the Greek meaning ‘active on the mind’. Properties such as the restoration of memory, alertness and mental lucidity are attributed to these pyrrolidonic substances, but the physiology of their activity is not known. The drug Piracetam (2-oxopyrrolidin acetamide) also belongs to this group. It is known that pyrrolidonic structures interfere with metabotropic glutamate receptors. In fact, over-the-counter preparations recommended in treatments to support mental functions are available on the market.

Because of its structure, PCA is hydrophilic and is present in the hydrolipidic film of the skin for the purposes of perspiration control, with consequent control of the presence of water in the tissues underlying the horny layer. And water, as is well known, is fundamental to preserving skin tone and elasticity.

Antimicrobial activity

A study to assess the antibacterial activity of PCA esters (Figure 2) was planned, prompted by the capacity for pyrrolidonic structures to interfere in a number of vital processes.

The minimum inhibitory concentration (MIC) of different PCA esters was measured against a range of bacteria, yeasts and fungi, and subsequently a Challenge Test with cosmetic preparations was performed

Bacteriostatic activity

The bacteriostatic activity of individual esters was determined in cultures of bacteria, yeasts and fungi (106 CFU/ml) belonging to standard ATCC collections in Tryptic Soy Broth, assessing the turbidity level of the inocula, compared with the control broth. Incubation was for 24 hours at 35°C.

The following strains were used:

• Staphylococcus aureus ATCC 6538;
• Pseudomonas aeruginosa ATCC 9027;
• Candida albicans ATCC 10231;
• Escherichia coli ATCC 8739;
• Aspergillus niger 16404.

Screening was carried out on esters of homologous families; results are expressed as MICs (Table 1).

Search for synergies

In order to improve and broaden the bacteriostatic activity spectrum of PCA esters with benzene rings, 1:1 mixtures of the esters combined with with phenoxyethanol were assessed (Table 4).

The bacteriostatic activity of the PCA benzylester and phenoxyethanol mixture increased with respect to bacteria and yeasts when compared with the activity of PCA benzylester alone, moreover its spectrum widened and it was also active against moulds.

Challenge test

Encouraged by these results, a challenge test was conducted using cosmetic emulsions containing the PCA benzylester and phenoxyethanol mixture at 0.5%. A decrease in micro-organisms was found in all the emulsions tested, in line with the criteria accepted in the European Union. Approximately, the decrease in micro-organisms was as follows:

Patch tests

To assess the potential irritant activity of the PCA benzylester and phenoxyethanol mixtures, 50% mixtures using the following diluents were prepared: diethylene glycol, monoethyl ether, propylene glycol, glycerine, butylene glycol and methyl propandiol. Patch tests on volunteers using a 2% of the various mixtures in a cosmetic emulsion.showed no irritant effects.