GB1567307A - Salts of glycyrrhizinic acid - Google Patents

Description

(54) SALTS OF GLYCYRRHIZINIC ACID (71) We, UNILEVER LIMITED, a company organised under the laws of Great Britain, of Unilever House, Blackfriars, London E/C 4, England, do hereby declare the invention for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement:- The invention is concerned with compounds having a bactericidal effect and a sweet taste which can be used in cosmetic compositions, in particular for oral use. More particularly the invention is concerned with the chemical combination of quaternary ammonium compounds with glycyrrhizinic acid.

The quaternary ammonium compounds are classified as cationic compounds having a bactericidal effect. Those with long chains are surfactants and are used in haircare products, for cleaning in the food industries and in medicine as disinfectants.

Although, in certain formulations, when mixed with nonionics, they are barely toxic and barely irritating, their very bitter taste makes it difficult to apply them in products for oral use.

The object of this invention is to prepare novel germicidal compounds of only slight toxidty, only slight irritation and a sweet taste. Besides, the invention is particularly concerned with their use in products for the care of the mouth.

The novel compounds according to the invention are obtained by neutralisation of glycyrrhizinic acid by one, two or three molecules of a quaternary ammonium base.

They are represented by the following general formula:

in which R1, R2 and R, are each hydrogen or ammonium or a quaternary ammonium cation having the structure

in which R4, R5 and Rt are each a C,, straight or branched chain, saturated or un saturated acyclic hydrocarbon radical, or two or three of R4, R5 and R6 together with the adjacent nitrogen atom represent a pyridine, piperidine or morpholine radical, and R is an alkyl, hydroxyalkyl, aryl, aralkyl, cyclohexyl or hydroxycyclohexyl radical, with the proviso that at least one of R1, R2 and R3 is a quaternary ammonium cation having the above structure and not more than one of the radicals R1-R, is ammonium.

These compounds are obtained by reaction of quaternary ammonium hydroxide with the mono-ammonium salt of glycyrrhizinic acid. The quaternary ammonium hydroxides are prepared in the usual way starting from halogenides. The monoammonium salt of glycyrrhizinic acid is a commercial product, named Glycamil, marketed by the firm of Inverni della Beffa, Milan.

Typical examples of quaternary ammonium compounds suitable for the synthesis of the compounds of the invention are the alkyl trimethyl ammonium halogenides of formula R-N(CH3)nX and the alkyl benzyl dimethyl ammonium halogenides of the formula R-N(CH,). . CH2CGHo . X, in which X represents chlorine, bromine or fluorine and R represents an alkyl radical with 8 to 18 carbon atoms. Other suitable compounds are the alkyl dimethyl (substituted benzyl) ammonium halogenides, the benzyl radical of which has as substituents one or more alkyl groups of 1 to 4 carbon atoms, such as a methyl radical, two methyl radicals, or an ethyl radical.

Various other quaternary ammonium compounds are also suitable, e.g. N-alkyl pyridinium halogenides and N-trialkyl hydroxycyclohexyl ammonium halogenides.

Quaternary ammonium compounds suitable for the preparation of the glycyrrhizinic acid compounds are, e.g. tetradecyl trimethyl ammonium bromide, hexadecyl trimethyl ammonium bromide and benzyl dimethyl alkyl ammonium halogenides such as benzyl dimethyl tetradecyl ammonium chloride.

Evaluation of bactericidal and anti-plaque activity The bactericidal efficiency can be evaluated by the minimum inhibitory concentration test, in which the lowest concentration of the microbiologically active compound is determined which inhibits the growth of the microorganisms chosen. This is a technique which is known to those skilled in the art.

Test tubes are prepared containing 9 ml of Jordan medium with 1) 2% sucrose, 2) blood serum and 3) 0.4% mucin. The compound of the invention is added at an appropriate concentration to the test tubes. The tubes are then inoculated with 0.2 ml of a twenty-four-hour-old culture of Streptococcus Ingbritt 00101, after which they are incubated at 379C for 48 hours and the evolution of the culture is observed. The results of the tests on four compounds according to the invention, to wit: tetradecyl trimethyl ammonium glycyrrhizinate, N-(tetradecyl)-pyridinium glycyrrhizinate, dodecyl dimethyl naphthylmethyl ammonium glycyrrhizinate and tetradecyl dimethyl benzyl ammonium glycyrrhizinate, compared with the tetradecyl trimethyl ammonium bromide taken as reference, are illustrated by the figures of Table I.

TABLE I Minimum inhibitory concentration

Media Jordan Jordan + serum Jordan + mucin (%) (%) (%) Tetradecyl trimethyl ammonium bromide < 0.0003 0.006 0.006 Tetradecyl trimethyl ammonium glycyrrhizinate 0.0006 0.006 0.003 N-(Tetradecyl)-pyridinium glycyrrhizinate 0.003 0.03 0.03 Dodecyl dimethyl naphthylmethyl ammonium glycyrrhizinate 0.03 > 0.06 0.06 Tetradecyl dimethyl benzyl ammonium glycyrrhizinate 0.003 0.03 0.03 From Table I it appears that the bactericidal activity of the compounds according to the invention is generally weaker than that of tetradecyl trimethyl ammonium bromide used as reference. This activity is completely satisfactory and sufficient for their use in products for the care of the mouth.

The bactericidal efficiency of the compounds according to the invention can also be evaluated by means of the biophotometer method. With this instrument, the growth curve of a Streptococcus strain (4M4) was followed. In the experiment described here, mono-, di- or tri(tetradecyl dimethyl benzylammonium) glycyrrhizinates were used at a concentration of 2 g per ml of culture medium.

An example of a series of growth curves produced when using this instrument is reproduced in the Figure.

In this figure, the abscissa represents the turbidity of a liquid culture medium con- taining growing bacterial cells, whereas the ordinate represents the passage of time during the experiment.

It will be noted that the control sample grew rapidly after innoculation and reached a maximum as measured turbidimetrically after about four hours.

The growth rate of the sample containing the mono quaternary ammonium salt of glycyrrhizinic acid was only slightly slower than that of the control, probably because this particular salt is only slightly soluble in water.

The growth rates of both of the other samples containing respectively the corresponding di-substituted and tri-substituted quaternary ammonium salts of glycyrrhizinate were both very low and did not reach a maximum value until after 36 hours indicating a strong bacteriostatic effect in both cases.

Examination of the curves also yields evidence that the growth inhibition of the cultures is correlated with the benzalkonium moiety of the molecule: 3.5% for the mono, 4.8% for the di and 5.5% for the tri-salt.

The compounds according to the invention were introduced into several types of toothpaste.

The minimum inhibitory concentrations (MIC) were obtained using aqueous extracts of the toothpastes. Experimental data showed that the bactericidal activity of the compounds according to the invention is the same, at equal concentration, whether in aqueous solution or in a toothpaste, provided the latter is adequately formulated.

For example, the MIC of an aluminium hydroxide toothpaste with 1% di(tetraethyl dimethyl benzylammonium) glycyrrhizinate equals that of a 1% aqueous solution of the same compound.

The bactericidal activity remains unaffected after 6 months' storage of the toothpaste at room temperature or at 40"C.

In vivo tests of anti-plaque activity were carried out in a panel of 7 human subjects.

The experiment reported here involved an aluminium hydroxide toothpaste containing 1% of di(tetradecyl dimethyl benzylammonium) glycyrrhizinate that was tested against a placebo and a positive control. The latter was a toothpaste containing 0.8% of Hibitane digluconate whose anti-plaque properties are well known but which in a short time produces brownish yellow stains on the teeth.

Anti-plaque activity was assessed over a period of 16 hours. A 0.5% aqueous erythrosin solution was used as disclosing agent.

The results of the statistical analysis (analysis of variance and Duncan's Multiple Range Test) show a significant difference between the three toothpastes, the confidence level being 95%. The Hibitane toothpaste is significantly more active than the paste containing the compound according to the invention, which is significantly more active than the placebo.

Organoleptic Tests The organoleptic tests of the four compounds mentioned before were carried out in aqueous or hydroalcoholic solutions of 0.2% by weight. The bitter and sweet tastes were compared with the taste of tetradecyl trimethyl ammonium bromide and evaluated by experts who noted the times during which the tastes were perceived. The results are indicated in Table II.

TABLE II Organoleptic evaluation

Taste Bitter Sweet Tetradecyl trimethyl ammonium bromide Extremely strong for No sweet taste 1 minute. whatsoever.

Strong for 4 minutes.

Weakens after 5 minutes.

Tetradecyl trimethyl ammonium glycyrrhizinate 1st minute: strong. First 9 minutes: strong.

Weakens after 2 minutes. Medium: 15 minutes.

Weakens after 20 minutes.

N-(Tetradecyl)-pyridinium glycyrrhizinate 1st minute: rather strong. First 7 minutes: strong.

Weakens after 3 minutes. Medium: 15 minutes.

Weakens after 25 minutes.

Dodecyl dimethyl naphthylmethyl ammonium First 2 minutes: strong. Medium: 12 minutes. glycyrrhizinate Weakens after 5 minutes. Weak: 15 minutes.

Weakens after 22 minutes.

Tetradecyl dimethyl benzyl ammonium First 30 seconds: medium. Strong: 4 minutes. glycyrrhizinate Weakens after 2 minutes. Medium: 15 minutes.

Weakens after 20 minutes.

It was observed that the compounds developed a bitter, less intense and less prolonged taste than that of the reference and which is in the case of the compounds according to the invention masked very quickly by an intense and persistens sweet taste.

Skin Irritation Test The irritation on the skin was tested on rabbits by means of the Liggett subcutaneous injection method. In this test solutions containing 0.5 to 0.01% by weight of the compound to be examined were injected at different places into the shaven skin of several laboratory rabbits (in general 4 for each compound). After 2448, or 72 hours if there was no reaction, the irritation of the skin was determined and expressed as a number. The following reactions were noted: a) erythema, b) oedema, c) dryness, d) cracking, e) peeling. The numerical value corresponding to each case is a) 0.15 (doubtful reaction), b) 0.25 (very slight reaction), c) 0.5 (slight reaction), d) 1 (visible reaction), e) 2 (distinct reaction) and 3 (very serious reaction).

The determination of the irritation of the compounds submitted to the test was made parallel with Vitamin C, 0.5% of which compound is taken as reference. The results are expressed in the ratio: Irritation due to the compound R -- Irritation due to Vitamin C TABLE III Cutaneous irritation

R Tetradecyl trimethyl ammonium bromide 1.16 Tetradecyl trimethyl ammonium glycyrrhizinate 0.75 N-(Tetradecyl)-pyridinium glycyrrhizinate 0.80 Dodecyl dimethyl naphthylmethyl ammonium glycyrrhizinate * Tetradecyl dimethyl benzyl ammonium glycyrrhi zinate 0.49 * Solubility v 0.5: R not determined.

The Table shows that the irritation caused by the reference is distinctly greater than that caused by the quaternary ammonium glycyrrhizinates.

The compounds according to the invention can be used in an effective and sufficient quantity in products in which a bactericidal effect is desired, more particularly in products in which little toxicity, little irritation and a sweet taste are desired.

As examples illustrating the compositions in which the compounds of the invention can be incorporated, can be cited: a) Products for the care of the mouth such as toothpastes, mouthwashes, dentifrices in powder and solid form, toothpicks, lipsticks. b) Medical products such as gargles, nose-drops. c) Various compositions such as adhesive compositions for envelopes, postage stamps, labels, foods.

It stands to reason that according to the above-mentioned description the compounds according to the invention and their mixtures can be used in a large number of products that are ingested, consumed or otherwise organoleptically perceived.

The quantity of the compounds of the invention which can be incorporated into such compositions can be adjusted to suit individual requirements. However, by way of example we can mention that of the products for the care of the mouth, it is preferable to employ by weight from 0.05 to 5.0% of the compounds in mouthwashes at a ready for use dilution and similarly from 0.5 to 5.0% in toothpastes or dental creams and from 0.5 to 10% in toothpowders.

It will be appreciated that the compounds of the invention, prepared for example according to the method described hereinbefore, can be the appropriate mono- or dior tri-quaternary ammonium derivatives. Accordingly, it is possible to incorporate in compositions, such as those referred to herein by way of example, any one of these three derivatives or a mixture of any two or indeed all three. It follows that the average degree of substitution by the quatemary ammonium in the molecule of the compound can be any value intermediate 1 and 3, that is intermediate the mono- and the tri-substituted derivatives, depending on the proportion of each which is present in the mixture. For example, a mixture containing equal proportions of the mono and the di substituted compound will have an average degree of substitution of 1.5.

Preferably the degree of substitution is from 1.5 to 2.5, most preferably 2.0, since we have discovered that the mono substituent with a degree of substitution of 1.0) is only slightly soluble in water and therefore is unlikely to have a significantly beneficial effect in products such as dentifrices. Also we have found that the tri-substituent (with a degree of substitution of 3.0) is too basic for use on its own in this type of product.

The following Examples are given to illustrate embodiments of the invention. It goes without saying that the Examples are merely given for illustrative purposes without, however, being limited thereto.

Unless otherwise indicated, all parts, percentages, ratios and proportions given in the present Specification are by weight.

EXAMPLE I.

Preparation of tetradecyl trimethyl ammonium glycyrrhizinate Preparation of tetradecyl trimethyl ammonium hydroxide 287 g (0.85 mole) tetradecyl trimethyl ammonium bromide (Noramium M 14 sold by the firm of Auby-Prochinor) are dissolved in 11 pure ethanol; 450 ml of a 1.85 N solution of potassium hydroxide are added to pure ethanol. (NORAMIUM is a trade mark). The mixture is allowed to stand for some hours so that the precipitation of the potassium bromide is complete, after which it is removed by filtration.

Preparation of glycyrrhizinate 50 g ammonium glycyrrhizinate (Glycamil) are suspended in 100 ml pure ethyl alcohol, after which 185 ml of a 0.644 N alcoholic solution of tetradecyl trimethyl ammonium hydroxide are added. Dissolution is complete.

This solution is then evaporated. In this way 72 g of a light-brown powder are obtained which softens and decomposes without melting at 2000 C.

UV = E mole A 240 = 8.350.

EXAMPLE II.

Preparation of N- (tetradecyl) -pyridinium glycyrrhizinate Preparation of N-(tetradecyl)-pyridinium hydroxide (= myristylpyridinium hydroxide) A column of a Dowex AG2-X8 resin, diameter of the column 24 mm, height 100 mm, volume 45 ml, i.e. an exchange capacity of about 63 meq. is preferred.

(DOWEX is a trade mark). This resin is then hydroxylated by passing two times through a 500 ml solution of 2 N potassium hydroxide, followed by rinsing with distilled water until neutrality is achieved. The column is then rinsed with 250 ml ethyl alcohol.

6 g myristylpyridinium bromide (16.8 meq), sold by the firm of Schuchardt, are dissolved in 20 ml ethyl alcohol awl.9 placed in the head of the column. The mixture is then eluted by four times 50 ml ethanol. The eluates are collected and completed to 250 ml.

Preparation of glycyrrhizinate 8.4 g (0.01 mole) ammonium glycyrrhizinate (Glycamil )are suspended in 20 ml pure ethyl alcohol; then 30 ml of a 0.067 N solution of myristylpyridinium hydroxide are added, after which dissolution occurs. This solution is then evaporated under vacuum.

Analysis: The purity is determined by titration of the bound quaternary ammonium, expressed in milli-equivalent for 100 g product.

Found 156.

Calculated 146.

EXAMPLE III.

Preparation of dodecyl dimethyl naphthylmethyl ammonium glycyrrhizinate Preparation of alkyl dimethyl naphthylmethyl ammonium hydroxide Alkyl dimethyl naphthylmethyl ammonium hydroxide is prepared from the corresponding chloride (BTC 1100) supplied by Noury-Onyx NV. 98% of the alkyl chain of BTC 1100 is dodecyl.

The hydroxide of BTC 1100 is obtained in alcoholic solution by ion exchange starting from BTC 1100 by passing over a previously hydroxylated Dowex AG2-X8 column, such as described in Example II.

Preparation of glycyrrhizinate 8.4 g (0.01 mole) Glycamil are suspended in 20 ml pure ethyl alcohol; then 30.5 ml of a 0.066 N solution of hydroxide of BTC 1100 are added, after which dissolution occurs. This solution is then evaporated under vacuum.

Analysis: The purity is determined by titration of the bound quaternary ammonium, expressed in milli-equivalent for 100 g product.

Found 130.2.

Calculated 130.4.

EXAMPLE IV.

Preparation of tetradecyl dimethyl benzyl ammonium glycyrrhizinate Preparation of alkyl dimethyl benzyl ammonium hydroxide Alkyl di methyl benzyl ammonium hydroxide is prepared from the corresponding chloride (BTC 824) supplied by Noury-Onyx NV. 93% of the alkyl chain of BTC 824 is tetradecyl.

The hydroxide of BTC 824 is obtained in alcoholic solution by ion exchange starting from BTC 824 by passing over a previously hydroxylated Dowex AG2--X8 column as hereinbefore described.

Preparation of glycyrrhizinate 8.4 g (0.01 mole) Glycamil are suspended in 20 ml pure ethyl alcohol; then 30.5 ml of a 0.066 N solution of BTC 824 hydroxide are added, after which dissolution occurs. This solution is evaporated under vacuum.

Analysis: The purity is determined by titration of the bound quaternary ammonium, expressed in milli-equivalent for 100 g product.

Found 145.5.

Calculated 134.8.

EXAMPLE V.

Delltitric$ /r Alumina 43 Water 22.5 Hydroxyethyl cellulose 1.3 Glycerine 15 Sorbitol (90%) 16 Tween 20 (TWEEN is a trade mark) 0.5 Flavour 1.2 Tetradecyl trimethyl ammonium glycyrrhizinate 0.5 EXAMPLE VI.

Dentifrice Calcium carbonate 20 Dicalcium phosphate 28 Sorbitil (70%) 20 Hydroxyethyl cellulose 1.5 Tween 20 0.5 Flavour 1.1 Dodecyl dimethyl naphthylmethyl ammonium glycyrrhizinate 0.5 Water 28.4 EXAMPLE VII.

Dentifrice Dicalcium phosphate 3W Hydroxyethyl cellulose 1.3 Sorbitol (70%) 10 Glycerine 5 Tetradecyl trimethyl ammonium glycyrrhizinate 0.5 Tween 60 0.5 Flavour 1.2 Water 31.5 EXAMPLE VIII.

Mouthwash Tetradecyl dimethyl benzyl ammonium glycyrrhizinate 0.1 Sorbitol 20 Distilled water 49.499 Tween 60 0.3 Bulgarian mint oil 0.05 Ceylon cinnamon oil 0.05 Red No. 2 (FD & C) 0.001 Alcohol BG 95"C 30 EXAMPLE IX.

Dentifrice % Tetradecyl dimethyl benzyl ammonium glycyrrhizinate 1.0 Humectant 27.0 Titanium dioxide 1.0 Emulsifier 1.5 Formalin (30%) 0.5 Alumina 55.0 Thickener 1.3 Water to 100 EXAMPLE X.

Dentifrice Tetradecyl dimethyl benzyl ammonium glycyrrhizinate 1.0 Humectant 27.0 Titanium dioxide 0.5 Emulsifier 1.5 Formalin (30%) 0.5 Alumina 50.0 Thickener 1.5 Water to 100

Claims (2)

1. WHAT WE CLAIM IS:1. Compounds represented by the general formula:

   in which R1, R2 and R3 are each hydrogen or ammonium or a quaternary ammonium cation having the structure:

   in which R, R5 and RG are each a C1 30 straight or branched chain, saturated or un saturated acyclic hydrocarbon radical, or two or three of R4, Rb and R6 together with the adjacent nitrogen atom represent a pyridine, piperidine or morpholine radical, and R; is an alkyl, hydroxyalkyl, aryl, aralkyl, cyclohexyl or hydroxycyclohexyl radical, with the proviso that at least one of R1, R2 and R, is a quaternary ammonium cation having the above structure and not more than one of the radicals R1-R3 is ammonium.
2. 2. A product for the care of the mouth comprising a compound according to claim 1 together with a physiologically acceptable diluent.