US3745222A - Cinnamyl phenols useful as antimicrobial agents - Google Patents

Abstract

SUBSTANCES WHICH ARE SUBJECT TO MICROBIAL SPOILAGE ARE PRESERVED BY ADDITION OF CINNAMYL PENOLS, E.G., 2CINNAMYLPHENOL, 4-CINNAMYL-PHENYL, 2-CINNAMYL-4-METHYLPHENOL, ETC.

Description

United States Patent ABSTRACT OF THE DISCLOSURE Substances which are subject to microbial spoilage are preserved by addition of cinnamyl phenols, e.g., 2-

cinnamylphenol, 4-cinnamyl-phenol, 2-cinnamyl-4-methylphenol, etc.

A non-exclusive, irrevocable, royalty-free license in the invention herein described, throughout the world for all purposes of the United States Government, with the power to grant sublicenses for such purposes, is hereby granted to the Government of the United States of America.

This invention relates to the preservation of substances which are normally subject to microbial spoilage. The objects of the invention include the provision of novel processes and compositions for accomplishing such preservation. Further objects of the invention will be evident from the following disclosure wherein parts and percentages are by weight unless otherwise specified. The abbrevia tion p.p.m. used herein refers to parts per million. Temperatures are given in degrees centigr-ade. The symbol 5 is used herein to designate the phenyl radical.

In accordance with the invention it has been found that certain agents exhibit unexpected antimicrobial activity and are useful for preserving all kinds of materials which are normally subject to microbial spoilage. The agents in question are certain cinnamylphenols, and their antimicrobial activity is unusual and unexpected because it is not shared by closely-related phenol derivatives.

Generically, the agents in accordance with the invention have the structure where at least one but not more than three of the Rs are hydroxy radicals, and the remainder of the Rs are each independently selected from the group consisting of lower alkyl, lower alkoxy, and hydrogen.

The compounds of the invention are especially useful because they are active against many microorganisms in the categories of bacteria, yeasts, and molds. In other words the compounds are not just active against one or few organisms; rather, they display broad-spectrum antimicrobial activity.

Examples of particular compounds within the scope of the invention are given below by way of illustration and not limitation:

4-Clnnamy1- OH lphenol (23,150 known as obtusastyrene) ice 2-Cinnamylphenol 2-Clnnamy1-4- methyl-phenol 2-Clnnamyl-4- methoxy-phenol Hg-CkCH-dv CHr-CH=CH CHQO 4-Clnnamyl-pryogallol The compounds of the invention display activities which are equal or even superior to those of widely-used antimicrobial agents. This is illustrated by the following: In general, the compounds of the invention are superior to such known agents as phenol, resorcinol, o-phenyl-phenol, and the alkali metal sorbates and benzoates. For instance, compounds of the invention, at concentrations of 12 to 25 p.p.m., were found to inhibit the growth of four bacteria (Bacillus cereus, Sarcina lwtea, Staphylococcus aureus, and Streptococcus lactis) whereas to achieve the same effect it required to 200 p.p.m. of o-phenylphenol, more than 800 p.p.m. of potassium sorbate,'and more than 1000 p.p.m. of sodium benzoate.

Alkyl 4-hydroxybenzoates are well known to exhibit potent microbial activity. In general, the compounds of the invention display a superior activity as compared to these benzoates wherein the alkyl group contains less than seven Patented July 10, 1973 v carbon atoms. For instance, whereas the compounds of the invention at a concentration of 12 to 25 p.p.m. will inhibit the growth of the four bacteria noted above, it requires concentrations of 100 to 400 p.p.m. for the same result to be achieved with the C -C alkyl 4-hydroxybenzoates. Moreover, it may be noted that the compounds of the invention have an advantage in that their watersolubility is higher than that of the higher alkyl (e.g., heptyl) 4-hydroxybenzoates, and that the compounds of the invention do not exhibit the strong and unpleasant odor which characterizes the said benzoates. Also, with respect to some microorganisms, the compounds of the invention inhibit growth when applied at levels less than required with the higher alkyl 4-hydroxybenzoates. This is the case, for example, with bacteria such as Acaligenes faecalis and E. coli, yeasts such as Pichia chodati, Hansenula anomala, and Saccharomyces cerevisiae, and molds including Aspergillus flavus, A. niger, P nicillium chrysogenum, Rhizopus semi, Botrytis cinerea, Byssochlttmys fulva, and Alternaria sp.

Of the various compounds included within the scope of the invention, the cinnamyl-substituted monophenols display particularly high antibiotic activity against a large .variety of different microorganisms, and therefore are preferred with respect to the cinnamyl derivatives of dior tri-phenols. Coming into special consideration is 4-cinnamyl-phenol because it displays antibiotic activity over a wide range of pH. The cinnamyl derivatives of monophenols are also preferred because they are essentially colorless, whereas those derived from dior tri-phenols exhibit various shades varying from red to brown.

As evident from the explanation immediately following Formula I above, the invention includes compounds wherein there may be nuclear lower alkyl or lower alkoxy substituents in addition to the hydroxy groups. In general, the compounds are preferred wherein such additional substituents are absent or, if present, are in small number, e.g., a total of one to two lower alkyl and/ or lower alkoxy substituents. Taking the foregoing facts into consideration, we prefer to employ the compounds of the sub-generic category represented by the formula wherein R is lower alkyl,

R" is lower alkoxy,

n is an integer from 0 to 2,

m is an integer from 0 to 2, and

the sum of n and m is not more than 2.

The invention encompasses not only the use of any of the above-described agents individually, but also mixtures thereof.

In preserving substances in accordance with the invention, any of the aforesaid agents or mixtures thereof are incorporated with the substance, using an amount of the agent to inhibit microbial growth. Additional conventional treatments such as dehydration, canning, refrigeraton, or freezing may be applied to the substance containing the added agent. The incorporation of the agent with the substance may involve a mixing of the substance and the agent-this is especially suitable where the substance is in liquid or particulate form. Where the substance is in the form of pieces of large dimensions, the agent may be incorporated therewith by coating it on the surface of the pieces. For such purpose the agent is preferably dispersed in a carrier-a liquid such as water, alcohol, wateralcohol blends, oils, or a finely-divided solid such as salt, starch, talc, or the like.

The invention is of wide versatility and can be applied for the preservation of all kinds of substances which are normally subject to microbial spoilage. Typical examples of such substances are listed below by way of example. Foodstuffs such as fruits, vegetables, juices, milk, eggs, meat, fish, grains, cereal products, cheese etc. Animal glues and rnucilages; dextrins; starch pastes and solutions; cosmetic, medicinal, and dental preparations; vitamin preparations; pastes, solutions, or other preparation of natural gums such as tragacanth, Arabic, acacia, karaya, locust bean, agar-agar, pectin, algin, etc.; fermentation broths, mashes, and residues from fermentation processes; whey; wines and Vinegars; animal feeds and ingredients of animal feeds such as fish meal, blood meals, feather meal, meat scraps, bone meal, tankage, grains, and oil-seed meals; proteins and protein hydrolysates; textile printing pastes; paints containing proteins or other spoilable dispersing agents; solutions of bark extracts or other tanning agents; molasses; by-products or wastes that contain potentially valuable carbohydrate, proteinous or fat ingredients such as stick liquor, corn steep liquor, fruit cannery wastes, citrus peels, cull fruit and vegetables, tops of root vegetables, distillers slops, pulp liquors, wash water from textile de-sizing operations, waste liquors from wool scouring plants, dairy and slaughter house wastes and liquors, etc.

The compounds of the invention may be synthesized by known procedures-for example, those disclosed by Hurd et al., Jour. Am. Chem. Soc., vol. 59, pp. 107-109; Barnes et al., Tetrahedron, vol. 21, pp. 2707-2715; Jurd, Experientia, vol. 24, pp. 858-860; Iurd, Tetrahedron, vol. 25, pp. l407-1416; and Jurd, Tetrahedron Letters No. 33, pp. 2863-2866. Typically, these syntheses involve condensing a selected phenol (phenol itself, a cresol, resorcinol, guaiacol, hydroquinone monomethyl ether, etc.) with cinnamyl bromide or cinnamyl alcohol.

The invention is further demonstrated by the following illustrative examples. For comparative purposes, various compounds, including some of known antimicrobial activity, are included in the reported experiments.

EXAMPLE 1 A series of compounds were assayed for effectiveness against bacteria, molds, and yeasts, using the following test procedure.

All compounds were tested at a concentration of 500 ppm. (w./v.) Plates were prepared by adding a measured amount of the candidate compound (in an appropriate solvent, i.e., acetone, ethanol, or water) to 10 ml. of sterilized medium, mixing thoroughly, pouring into 60 x 15 mm. Petri dishes, and allowing the gel to set. The plates were then inoculated with the test organisms. In the case of bacteria and yeasts the inoculation was done by Lederbergs replica plating technique, applying nine bacteria or seven yeasts on each plate. In the case of molds, drops of homogenized culture were placed on the surface of the plates, applying three to four molds per plate.

The media used were: plate count agar (Difco) for bacteria; potato dextrose agar (Difco) for yeasts and molds.

Control plates were also prepared containing the media plus the same solvent used for the candidate agents and inoculated with the same organisms.

The plates were incubated at 28 C. for one to five days and evaluated by comparison with the controls.

The results are expressed on the basis:

+ for effective to inhibit growth; 2 for not completely effective; faint growth occurs for ineffective; growth occurs The results are summarized in the following tables.

TABLE I [Activity of various compounds (at cone. of 500 ppm.) against bacteria] 2-cin- 2-cin- 4-cini-cin- 4-cinnamyl-5- namyl-5- Cinnamylnamylnamylmethoxymethoxynamyl- Methoxyphenol resoreinol pyrogallol quinol quinone eugenol Phenol gallol qulnol Bacillus cereas Sarcina Zatca Staphylococcus aureas Streptococcus lactz's Acaligeaes faecalis Escherichia coli :l: Pseudomo'nas aeragz'nosa Salmonella typhimuriam :i: Scrratia marcesceas :l: :1:

TABLE II [Activity of various compounds (at cone. of 500 ppm.) against yeasts] 2cin- 2-cini-cln- -oin- 4-cinnamy1-5- namy1-5- Cinnamylnamylnamylmethoxymethoxynamylphenol resorcinol pyrogallol quinol quinone eugenol Zygosacchomyces japonicusun. Candida tropicalis Pichia chodati Hansenala anomala Sancharomyces cerem'siae Saccharomyces mellis Torala atilis Hansenula melliqai- Candida chalmersL. Saccharomyces rosei Zygosaccharomyccs barke1i TABLE III [Activity of various compounds (at cone. of 500 ppm.) against molds] 2-ein- 2-oin- 4-cin- 4-cin- 4-cinnamyl-E- namyl-5- Cinnamylnamylnamylmethoxymethoxynamyl- Resor- Pyro- Methoxyphenol resorcinol pyrogallol qmnol qumone eugenol Phenol cinol gallol quinol Aspergillas flazms =1: Aspergillus m'ycr d: Penicillium chrysogcnam Rhizopus sent; Botrytis cinerea... Byssochlamya julvai Alternaria sp EXAMPLE 2 1, except that in this case the assays were conducted with varying amounts of each compound to determine the mini- A series of compounds were assayed for eifectiveness mum concentration thereof required to inhibit growth. against bacteria, molds, and yeasts as set forth in Example The results are set forth in the following tables.

TABLE IV [Minimal inhibitory concentrations (in p.p.m.) of various compounds against bacteria] 2-einnamy1- 2-methyl-4- 2-cinnamyl- 4-cinnamyl- 2-cinnamy1- 4-methoxyoinnamyl- 4-methylo-Phenyl- Potassium Sodium phenol phenol phenol phenol phenol phenol sorbate benzoate Bacillus cereus. 25 12 25 12 12 100-200 800 1, 000 Sarcina Zutea 25 25 25 12 12 200 800 1 000 Staphylococcus aa 25 25 25 12 12 800 1, 000 Sire tococc'us lactis-.- 25 12 25 12 12 00 800 1,000 Aw gemfaecam 50-100 200 200 200 200 100-200 800 1, 00o Escherichia coli 50-100 200 200 200 200 100-200 800 1, 000

TABLE V [Minimal inhibitory concentrations (in p.p.m.) oi various compounds against yeasts] 2-cinnamyl- 2-methyl-4- 2-cinnamyl- 4-cinnamy1- 2-cinnamyl- 4-methoxycinnamyla-methylo-Phenyl- Potassium Sodium phenol phenol phenol phenol phenol phenol sorbate benzoate Zygosaccharomyces japo nicus 12 12 2 1 1 100-200 800 1, 000 Candida tropicalis 12 12 50 25 100 200-400 250-500 Pichia chodati 25 200 200 200 100 800 1, 000 Hanse'aula anomala. 50 50 100 100 -200 800 1, 000 Saccharomyces cercvisiae 25 25 50 25 25 100-200 800 1, 000 Torula utilis 50 50 200 100 200 100-200 800 1 000 TABLE VI [Minimal inhibitory concentrations (in ppm.) of various compounds against molds] 2-cinnamy1- 2-methyl-4- 2-oinnamyl- A-cinnamyl- 2-cinnamyl- 4-methoxycinnamyl- 4-methy1- o-Phenyl- Potassium Sodium phenol phenol phenol phenol phenol phenol sorbate benzoate Aspergillus flaws 100 100 20 200 1 0 800 1, 000 Asperqillas nicer"-.- 50 100 200 200 200 100 800 1 000 Penicillium chryaogenam. 50 25-50 200 50 100 50 800 1, 000 Rhizopas semi 6 50 200 50 100 100 200 500 Botrytis cinerea 25-50 12-25 200 50 25 12-25 200 500 Byssochlamq s fulva- 25 25 50 25 25 50 800 1, 000 Alternaria sp 25 25 100 50 100 50 400 500 TABLE VII [Minimal inhibitory concentrations (in p.p.m.) of 4-cinnamyl-phenol and several a1kyl4-hydroxybenzoates against bacteria, yeasts, and molds] Heptyl 4- Amyl 4- n-Butyl 4- n-Propyl 4- Ethyl 4- Methyl 4- 4-einnamylhydroxyhydroxyhydroxyhydroxyhydroxyhydroxyphenol benxoate benzoate benzoate benzoate benzoate bemoate Bacillus cerws 25 12 100 200 400 200 200 Sarct'aa latea 25 12 100 200 400 200 200 f" 25 12 100 200 400 200 200 Stre 25 12 100 200 400 200 200 Acaligenes faecalis 50-100 200 200 200 400 200 200 Escherichia coli- 50 100 200 200 200 400 200 200 Zgg j 12 12-25 50 100 100-200 200 200 Candida tropicalis- 12 12-25 50 100 200 200 200 .Pz'chia chodati. 25 200 100 100 200-400 200 200 Hansenula anoma 50 200 100 100 200-400 200 200 Saccharomg/ces cerev 25 100 50 100 200 200 200 T l iili 50 25 100 100 200 200 200 Aspergillas flavus 100 200 100 200 200 200 200 Aspergillus niger 50 100 200 200 200 200 Penicilliam chi 0 200 100 100 200 200 200 Rhizopus 6 2 50 100 200 200 200 Botrytis cinerea 25-50 50-100 50 100 100-200 100 100 Byssochla'mys falva 2 0 100 100 200 200 200 Alternaria sp 50-100 50 50 100-200 100 200 EXAMPLE 3 inhibit growth at the particular pH.

The results are tabulated below.

and held at room temperature. The inoculated juices 25 fermentation (gas production) b for initiation of fermentation is at all.

TABLE VIII were observed at intervals to detect the time at which egan. This is a measure of microbial activity--where the organisms multiply freely, the time for initiation of fermentation is short; conversely, where microbial growth is inhibited the time long or does not occur [Effect of pH on minimal inhibitory conoehtration (p.p.m.) 0t i-oinnamyl-phenoi and 4-oinnamyl-resoroinol] 4-cinnamy1-phenol 4-cinnamyl-resorcinol pH7 p116 pH5 pH4 pH3 p117 pH6 p115 p114 pH 3 Bacillus cereus 25 25 25 200 100 6. 25 Sarcina lutea 25 25 25 100 25 f" 1 aareas. 25 25 25 100 50 25 Streptococcus lactis- 25 25 25 00 50 25 Acaligenes faecalis- 50 25 25 200 100 25 Escherichia colz',. 25 50 50 200 100 2 L jap 12.5 12.5 100 to so so Candida tropicalis- 25 25 50 50 50 Pithia 50 50 100 50 Hansemtla 1 50 50 100 100 100 75 Saccharomyces cerevz'siae 25 25 50 50 100 50 Geotriehum sp Torula utilis- I 50 50 75 50 75 50 Agpgrgillu3 flazmx 100 100 100 50 200 200 200 100 Aspergillus m'ger" 100 100 75 37. 5 200 200 100 100 Penicilliam chrusogenum 50 50 50 12- 5 100 100 50 50 Rhizopus sr'ntz' 25 25 25 12. 5 100 100 100 50 Botrz tis oinerm 25 25 25 12. 5 100 100 75 50 P W m. 25 25 25 12. a 100 100 75 50 Alternaria sp 5 12.5 75 50 The results are tabulated below.

TABLE IX [Influence oi 4-cinnamyl-phenoi on fermentation of fruit juice inoculated with yeasts] Time for initiation of fermentation, hours Amount of inoculum, Zero 12.5 p.p.m. 25 p.p.m. 50 p.p.m. 100 p.p.m. 200 p.p.m. Run Substrate Inoeulum cells/ml. 4-CP of 4-0? of 4-01 of 4-CP of 4-01 of 4-01? 1 Grape juice Wild yeast- 10 7 n.d. 12 n.d. 44 11,4 1.- (10.. do- 10 17 17 30 62 96 n.d. 2 do. Wine yeast 10 12 22 45 146 w m 4 do--- do.. 10 41 75 w on m 5 Apple juice. do 10 46 42 76 w m c NOTE.-4-CP designates 4-cinnamyl-phenol; n.d. means not determined; 00 means that fermentation did not occur; the organisms had been destroyed.

EXAMPLE 4 Having thus described the inve ntion, what is claimed is:

1. A process for inhibiting the growth of microorganisms selected from the group consisting of bacteria,

yeasts, and molds in a substance normally subject to cultures of wild yeast or pure wine yeast (S. cerevisz'ae) 7 spoilage by said organisms, which comprises applying to 9 10 said substance an effective microbial growth-inhibiting 6. The process of claim 1 wherein the compound is amount of a compound of the structure 4-cinnamyl-resorcinol.

7. The process of claim 1 wherein the compound is R 4-cinnamyl-pyroga1lo1. R R 5 References Cited R R Jurd: Tetrahedron Letters No. 33, pp. 2863-2866,

1969. HFCH=CH Jurd: Tetrahedron, vol. 25, pp. 1407-1416, 1969.

10 Jurd: Experientia, vol. 24, pp. 858-860, 1968.

wheren 1 to 3 of the Rs are hydroxy, and the remainder g et Chem Abst" 3396-3398 of the Rs are each independently selected from the group Mumm et aL: Chem. Ab VOL 32 pp 522 52 4 1938 Consisting oflowfir alkyl and hydrogeq' Hurd et al.: Jour. Am. Chem. Soc. vol. 59 pp. 107- 2. The process of claim 1 wherein the compound 1s 15 109 1937 4-cinr1amy1-pheno1. I

3. The process of claim 1 wherein the compound is gg et Tetrahedron 2707 2715 2-cinnamy1-phenol.

4' The process of claim 1 wherein the compound is Clarsen et al.. Chem. Abst., vol. 19, p. 2038, 1925. y y -p 20 JEROME D. GOLDBERG, Primary Examiner 5. The process of claim 1 wherein the compound is 2 cinnamy1 4 methy1 pheno1 A. J. ROBINSON, Assrstant Exarmner