US3810990A - Inhibiting growth of bacteria - Google Patents

Abstract

Substances which are subject to microbial spoilage are preserved by addition of certain compounds, e.g., 3-0-methyl-quercetin; a composition of 5-0-methyl- and 7-0-methylquercetin; 4- or 7methoxycoumarin; 2-(4-hydroxyphenyl)-benzofuran; or various mixtures of these.

Description

United States Patent [191 Jurd et a1.

' [451 May 14,1974

[ INHIBITING GROWTH OF BACTERIA [75] Inventors: Leonard Jurd, Berkeley; Alfred Douglas King, Jr., Martinez, both of Calif.

[73] Assignee: The United States of America as represented by the Secretary of Agriculture, Washington, DC.

22 Filed: Feb. 27, 1970 21 App1.No.:15,302

[52] US. Cl. .Q 424/283 [51] Int. Cl A0ln 9/28 [58] Field of Search 424/281, 283

[56] V References Cited OTHER PUBLICATIONS Chakaborty et al., Chem. Abst. Vol. 56 (1962) page DeGreef et a1., Chem. Abst. Vol. 70 (1969) page 353210.

Letan, Chem. Abst. Vol. 65 (1966) page 112421. Bandyukova et a1., Chem. Abst. V01. 60 (1964) page 8625b.

Primary Examiner-Sam Rosen Attorney, Agent, or Firm-M. Howard Silverstein; William Takacs 1 Claim, No Drawings 1 INHIBITING GROWTH OF BACTERIA A nonexclusive, irrevocable, royalty-free license in the invention herein described, throughout the world for all purposes of the United States Government, with fied. The abbreviation ppm used herein refers to parts per million. Temperatures are given in degrees Centigrade.

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 derivatives of quercetin, coumarin, and benzofuran, and their antimicrobial activity is totally unusual and unexpected because it is not shared by closely-related derivatives, including isomers. The agents having these important andtechnically-useful properties are next described in detail.

Certain derivatives of quercetin are included among the agents of the invention. Quercetin is a known compound having the structure:

One of the agents of the invention is the compound 3-0-methylquercetin. Its structure can be visualized from formula I wherein the substituent at the 3 position is -OCH rather than OH. It has been found that 3-0- methylquercetin is especially active against bacteria and in a series of tests it was shown to be effective against 12 out of a total of 13 different types of bacteria tested whereas many other quercetin ethers display litand 7-methoxycoumarin which has the structure These compounds are especially active against molds and yeasts. In a series of tests they were shown to be effective against 5 out of 5 molds tested, and against 6 to 7 out of 7 yeasts tested. On the other hand, many other derivatives-of coumarin were found to have little if any activity against molds or yeasts.

Another of the agents of the invention is 2-(4- hydroxyphenyl)-benzofuran which has the structure This compound is especially active against yeasts and is also active against some moldsAspergillus glaucus and a brown rot mold of the genus Sclerotinia, for example-and many bacteria. In a series of tests the compound in question was shown to be effective against 8 out of a total of 8 different yeasts, and against 8 out of a total of 13 different bacteria. Various other benzofuran derivatives were found to be virtually inactive against yeasts and bacteria.

The invention encompasses not only the use of any of the above-described agents individually, but also mixtures thereof. For example, where it is desired to inhibit the proliferation of molds and yeasts plus bacteria, one would employ a composite of (a) 3-0- methylquercetin or the mixture of S-O-methyland 70- methylquercetin plus (b) 4-methoxyor 7- methoxycoumarin. Further extensions of this principle will be obvious from the foregoing illustration. Particular examples of this phase of the invention include the following: A composition of (a) 3-Omethylquercetin and (b) a member of the group consisting of 4- methoxycoumarin and 7-methoxycoumarin. A composition of (a) 3-Omethylquercetin and (b) 2-(4- hydroxyphenyl)-benzofuran. A composition of (a) a mixture of 5-O-methylquercetin and 7-0- methylquercetin, and (b) a member of the group consisting of 4-methoxycoumarin and 7- methoxycoumarin. A composition of (a) a mixture of S-O-methylquercetin and 7-0-methylquercetin, and (b) 2-(4-hydroxyphenyl)-benzofuran.

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, refrigeration, 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 inliquid or particulate form. Where the substance is in the form of pieces of large dimensions the agent may be incorporated there with 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, water-alcohol blends, 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 mucilages; dextrins; starch pastes and solutions; cosmetic, medicinal, and dental preparations; vitamin preparations; pastes, solutions, or other preparations of natural gums such as tragacanth, Arabic, acacia, karaya, locust bean, agaragar, 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 meals, blood meals, feather meal, meat scraps, bone metal, 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 vegeta bles, 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 invention is further demonstrated by the following illustrative examples.

EXAMPLE 1 4 SYNTHESIS OF 3-0-METHYLQUERCET1N (METHOD A) a. Preparation of 5,7,3,4-tetra-O-benzoyl quercetin. Benzoyl chloride (80 ml.) was added in two portions during minutes to a solution of rutin g) and potassium carbonate (160 g) in water (1.5 1.). After 40 minutes the precipitated solid was collected and dissolved in boiling ethanol (300 ml). Concentrated hydrochloric acid (60 ml) was added and the clear solution was heated on a steam bath for 1 hour. Water (250 ml) was added. The oil which separated solidified on cooling. It was collected, dissolved in acetone, diluted with methanol and concentrated until most of the acetone had distilled. The crystalline solid which separated from the hot methanol solution was collected (8.4 g; m.p. 204-205). Recrystallized from acetonemethanol, the 5,7,3,4-tetrabenzoate separated as slightly yellow, granular crystals, m.p. 209 (7.8 g). If the tetrabenzoate is allowed to crystallize slowly from acetone alone it separates in cream-colored needles which melt at about l26130, resolidify and then melt at 209.

Calcd. for C H O C, 71.85; H, 3.65.

Found: C, 71.4; H, 3.71.

b. Preparation of 3-O-methyl-5,7,3',4-tetra-O- benzoyl quercetin. 5,7,3 ,4-Tetra-O-benzoyl quercetin (19.5 g) was methylated with dimethyl sulphate (25.0 ml), potassium carbonate (40 g) and acetone (500 ml) for 2 hours. The product, isolated in the usual way,

crystallized from ethyl acetate and from acetonemethanol as colorless needles, mp. 181 (16.8 g).

Calcd. for C H O C, 72.1; H, 3.85; 1 MeO-, 4.24.

Found: C, 71.7; H, 3.96; MeO 4.77.

c. Preparation of 3-0-methylquercetin. Potassium hydroxide (15 g) was added to a suspension of 11 g. of product b in warm methanol (100 ml). The resulting clear solution was diluted with water 100 ml), warmed for 5 minutes and acidified. The mixture was concentrated until most of the methanol had distilled. The yellow product was collected, washed with benzene and recrystallized from aqueous methanol. 3-0- Methylquercetinseparated as yellow needles, mp. 275 (lit. m.p. 273-275) (4.1 g).

Calcd. for C H O C, 60.75; H, 3.83; 1 MeO-, 9.87.

Found: C, 60.8; H, 3.90; MeO-, 9.73.

EXAMPLE 2 SYNTHESlS OF 3-0-METHYLQUERCET1N (METHOD B) 3 ,5-Dihydroxy-7-benzyloxy-3 ',4- diphenylmethylenedioxyflavone. A mixture of benzylquercetin (4.3 g) and a,a-dichlorodiphenylmethane (2.9 g; 1.1 mol. equivs.) was heated in an oilbath at 220 for 5 minutes, cooled and dissolved in acetone (50 ml). The solution was concentrated and diluted with methanol. The crystalline product (4.1 g) was recrystallized from acetonemethanol. 3.5- Dihydroxy-7-benzyloxy-3 ',4 -diphenylmethylenedioxyflavone was obtained as yellow granular crystals, m.p. 1

Calcd. for C H O C, 75.5; H, 4.35.

Found: C, 75.3; H, 4.32.

5-Hydroxy-3-methoxy-7-benzy1oxy-3 ',4 diphenylmethylenedioxyflavone. The 3,5-dihydroxy compound (1.4 g) was methylated with dimethyl sulphate (0.35 g; 1.1 mol. equivs.), potassium carbonate and acetone in the usual way. The product, 5-hydroxy- 3 -methoxy-7-benzyloxy-3 ',4 '-diphenylmethylenedioxyflavone, separated from acetone-methanol as yellow needles, mp. 170 (1.2 g). With alcoholic ferric chloride it gave an intense-green-brown color.

Calcd. for C H O C; 75.8; H, 4.60; MeO-, 5.44.

Found: C, 75.8; H, 4.57; MeO-, 5.46.

The above methyl ether (1.0 g) was heated on a steam bath for 30 minutes with glacial acetic acid 15.0 ml) and concentrated hydrochloric acid (15.0 ml). Water (400 ml) and benzene (50 ml) were added and the yellow solid was collected. Recrystallized from aqueous methanol, 3-0-methylquercetin, m.p. and mixed m.p. 275, was obtained (0.3 g).

EXAMPLE 3 SYNTHESIS OF S-O-METHYL- 7-0METHYLQUERCETIN 5-0-Methyl- 7-0-methylquercetin. A mixture of quercetin 3,7,3,4-tetraacetate (4.70 g), dimethyl sulfate (2.50 g), anhydrous potassium carbonate (5.0 g) and anhydrous acetone (50.0 ml) was heated under reflux for one hour and filtered. The acetone filtrate was evaporated to an oil..This was dissolved in percent ethanol (20.0 ml) and 50 percent aqueous sulfuric acid (5.0 ml) and the solution heated for one hour. Yellow crystals separated (1.0 g). These were collected and the filtrate, diluted with more water, was heated for a further two hours whereupon a second drop of crystals separated. The combined crystalline products were digested with methanol (20 ml) and water (40 ml) leaving a yellow solid (2.41 g). Silicic chromatography of the product showed that this solid consisted primarily of a mixture of the 5-0-methylquercetin and 7-0- methylquercetin. The S-O-methylquercetin constituent tin (60 g) in acetic anhydride (180 ml). After 4 minutes excess of water was added and the solid was collected. Recrystallized from acetone-methanol quercetin 3,7,3 ,4-tetraacetate separated as pale yellow needles, mp. l90-l 91 (70 g). These gave an intense red color with alcoholic ferric chloride.

EXAMPLE 4 SYNTHESIS OF 7-0-BENZOYLQUERCETIN Benzoyl chloride (25.0 ml) was added to a vigorously stirred solution ofrutin (20.0 g) and borax (30 g) in water (400 ml) at room temperature. After minutes precipitated benzoic acid was filtered and the filtrate was treated with concentrated hydrochloric acid (100 ml) and heated on a steam-bath for 30 minutes. The

EXAMPLE 5 i A lot of fresh skim milk was divided into two lO0-ml samples. Into one sample was incorporated sufficient 3- O-methyl quercetin to give a concentration thereof of 500 ppm. Each sample was inoculated by adding 1 ml of skim milk which had been allowed to stand at room temperature for several hours. The two samples were then held in an incubator at 28 C. for 3 days and examined at the end of this period. The control sample was found to be separated into curds and whey; the treated sample was still homogeneous.

EXAMPLE 6 A 'lot of grape juice was divided into two 100 ml samples. Into one sample was incorporated 2-(4-hydroxyphenyl)-benzofuran in a concentration of 500 ppm.

' Both samples were inoculated with wine yeast (Saccharomyces cerevisiae) at a concentration of about 2.5 X 10 cells per sample. The samples were then stored at 28 C. for several days.

On the third day it was observed that the contro juice was foamy-indicating that active fermentation. was taking place. The treated juice was not foaming at.

all.

- On the fourth day the two samples were assayed for yeast cell count. The following results were obtained:

Treated juice: 10 yeast cells/ml. Control juice: 7 -l0 yeast cells/ml.

EXAMPLE 7 A series of derivatives ofquercetin, coumarin, and benzofuran were assayed for effectiveness against bacteria, molds, and yeasts, using the following test procedure:

All compounds are tested at the concentration of 500 ppm (w/v) using premeasured amounts of agar containing the compound from test tubes or dispensing pipettes to measure 10 ml of sterile medium into 60 X 15 mm plastic Petri plates. Because of the relative insolubility of some of the compounds the weighed compound is mixed with gum tragacanth (0.2 percent) in a ground glass tissue homogenizer. A few ml. of water are added during grinding to aid in suspending the com pound in the agar medium prior to steam sterilizing 15 psig for 15 minutes). Plate count agar (Difco) (pH 7.3) or potato dextrose agar (Difco) (pH 5.6) are used for bacterial or yeast and mold culture, respectively.

The inoculum is prepared from 24- or 48-hour cultures of bacteria grown on tryptone glucose yeast ex tract broth, yeast grown on Sabouraud maltose broth, or fungi grown on potato dextrose agar. Turbidity measurements of bacterial and yeast suspensions are used as a means of estimating the number of cells per ml. of

broth. Dilutions in 0.1 percent peptone water are then made to give between 30 and 300 colonies per Petri plate. The mold cultures are scraped off the surface of the slant and macerated in a Waring blender for 1 min ute with ml of sterile peptone water. The solid medium in the Petri plate is inoculated by placing a loopful of the mold macerate in the center of the plate. Control plates contain medium, gum tragacanth, and the microorganism.

After incubation of 30 C. or 35 C. the results are evaluated after 48 hours and again after 1, 2, and 3 weeks. The bacteria and yeast data are determined by counting colonies under a 7 X wide field microscope. The results are expressed on the basis:

+ for effective to inhibit growth for ineffective, growth occurs In conducting the assays, each candidate agent was preliminarily tested for effectiveness against 1 to 3 microbial species, e.g., B. cereus, E. coli, and P. aeruginosa, in the case of tests for activity against bacteria. Where the agent exhibited little or no activity in this preliminary screening, no further assays were carried out therewith, except in a few cases as will be evident below. A similar plan was followed in testing the candidate agents against other. types of microorgan isms, using in the preliminary tests A. glaucus in the case of molds, and S. rouxii and S. mallis in the case of yeasts.

The results are summarized in the following tables.

Compound tested 4-methoxycoumarin 4-hydroxycoumarin 4-aliyloxycoumarin 4-benzoyloxycoumarin 7-methoxycoumarin 7-hydroxyc'oumarin 7-geranoxycoumarin 7-allyloxycoumarin 7-benzoyl0xyc0umarin S-methoxycoumarin 5-geranoxy-7- methoxycournarin 5,7-di-methoxycoumarin 5-hydroxy-7- methoxycoum arin 7-hydroxy-6- methoxycoumarin Compound tested @methoxycoumarin 4-hydroxycoumarin 4-allyloxycoumarin 4-benz0yi0xycoumarin 7-methoxycoumarin 7-hydroxycoumarin 7-geranoxycoumarin 7allyloxycoumarin 'methoxycoumarin 5-geranoxy-7- methoxycoumann 5,7-di-methoxycoumarin 5-hydroxy-7- methoxycoumarin 7-hydroxy6-methoxycoumarin Activity of Coumarin Derivatives against Molds Aspergillus glaucus Zygosacc haromyces J aponica var. soya TABLE 11 Aspergillus oryzae Aspargillus nlger Botrytis cinerea TABLE III Byssochlamys fulva Activity of Coumarin Derivatives against Yeasts saccharomyces barkeri Hanseniaspora melligeri Pichia chodati var. fermentans Candida chalmersi Candida tropicalis Sacchammyces rouxii charcmyces mallis llll+lll+ TABLE IV Activity of Bcnzofuran Derivatives against Yeasts Compound tested Zygosaccharomyces japonica var. soya Zygosaccharomyces barkeri Hanscniaspora melligeri Pichia chodati var. fermentans Candida chalmersi Candida tropicaiis Saccharomyces rouxii Sacchammyces mallis w 12 Certain of the subject ma tt erdisclosed herein, in particular the syntheses of quercetin derivatives, is the sole invention of Leonard J urd and is claimed in his copending application Ser. No. 18,314, filed Mar. 10, 1970, now U.S. Pat. No. 3,661,890.

Having thus described the invention, what is claimed is:

1. A method for inhibiting the growth of bacteria which comprises applying 3-0-methylquercetin thereto.

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