LOW CARIOGENIC SWEETENING AGENTS

Abstract: Hernandulcin, a sesquiterpene and related compounds possess a sweetness intensity about three orders of magnitude greater than that of sucrose. These compounds are non-mutagenic and non-toxic under conventional assay procedures. In addition to attractive utility in foods, beverages and pharmaceutical compositions, these compounds afford a low-cariogenic, essentially non-nutritive sweetening agent for inclusion in oral hygiene products.

Description: LOW CARIOGENIC SWEETENING AGENTS The invention was made with United States Government support under contract NO1-DE-0245, awarded by the National Institute of Dental Research, National Institutes of Health. It is generally agreed there continues to be a need for highly effective non-nutritive and non-cariogenic sweetening agents to replace the extensive use of sugar in the diets of most individ¬ uals. Synthetic substances such as saccharin, cyclamate, and asparta e have been or are being used in the United States for this purpose, while natural¬ ly occurring substances such as phyllodulcin, stevioside, and glycyrrhizin are used in Japan. None of these substances is ideal, however, either because of taste characteristics that are generally perceived as unpleasant, questionable safety, chemical in¬ stability, or because of the relatively high cost of production. Unfortunately, insufficient information exists on the molecular structures which are neces¬ sary to impart sweetness to molecules to allow scientists to design sweet-tasting molecules which can serve as prototypes for the commercial develop¬ ment of sweeter and safer compounds. Between 1570 and 1576, a Spanish physician by the name of Francisco Hernandez described a remark¬ able sweet plant known to the Aztecs as "Tzonpelic xihuitl" , the literal translation being "sweet herb". The accurate description and illustration of the plant in his writings indicated he was referring to Lippia dulcis Trev. , a plant which is native to central Mexico. The principal sweet component of this plant, which was present mainly in the leaves and flowers , was isolated in pure form after solvent partition and chromatographic fractionation. Using this component, which we named hernandulcin in honor of its Spanish discover, other compounds structurally related to hernandulcin were developed and studies. The sweetening agents of the present invention, all of which can be classified as sesquiterpenes, have the general formula (I) : in which RHIY is a lower alkyl of one to four carbon atoms, an alcohol such as CH_ CH 2 C00H; R. is hyrod hydrogen or a lower alky ■ *l of one to four carbon atoms; R_3 is a lower alkyl or alkenyl of one to five carbon atoms, including an oxygen-containing alkyl; and n is an integer from 1 to 8. More particularly, the present invention encompasses compounds of general formula I wherein: R, is -CH- -CH 2 CH 3 -(CH 2 ) 2 CH 3 -(CH 2 ) 3 CH 3 -CH 2 OH -CH 2 C00H is -H -CH 2 CH 3 -(CH 2 ) 2 CH 3 -(CH 2 ) 3 CH 3 ; and -R, is -CH 3 -CH 2 CH 3 -CH 2 CH=C(CH 3 ) 2 -CH 2 CH 2 CH(CH 3 ) 2 -CH 2 CH=CHCH 3 -CH(CH 3 ) (CH 2 OH) -CH(CH 3 ) (CH 2 COOH) The molecule of the compounds of general formula I are all characterized as possessing two adjacent asymmetric centers involving both a cycloaliphatic ring structure and an alkyl substituent. Thus, the agents may appear as both dextro and levo stereo- isomers, and in fact the process outlined in the examples yields a racemic mixture of the two isomers. If desired, of course, the mixture may be separated according to isomers by using conventional tech¬ niques. Exemplary of the compounds according to the present invention, hernandulcin, or 6-{l',5'- dimethyl-1'-hydroxyhex-4'-enyl)-3-methylcyclohex-2 enone, in its optically active form, is extremely sweet, being some 1,000 times as sweet as sucrose when compared on a molar basis (at 0.27 molar sucrose) and about 1,500 times as sweet when compared on a weight basis. The synthetic, racemic mixture of levo and dextro stereoisomers of hernandulcin like¬ wise possesses a very attractive sweetness intensity, about one-half that of the isolated natural product. The sweetness of hernandulcin is believed to be associated with asymmetry of the 6 and l 1 carbon atoms as well as with the proximity of the keto and hydroxyl groups. The taste properties related to the oxygen-containing groups of general formula I remain essentially unaffected by substitution of hydrogen or a lower alkyl group at the R~ position. Variation in substituents R. and R, can be effected to include alkyl or alkene groups and particularly hydroxyl or σarboxyl groups for increasing the water solubility of the sweetening agent. The hernandulcin molecule itself is soluble in water to the extent of about 015 gram per liter. While this is generally sufficient, because of the intense sweetness exhibited, modifica¬ tion of structure to improve water solubility in various foodstuff formulations is available as may be desired. For example, hydroxyl-containing sub¬ stituents at R- or R. positions may be further modified by well known conversions to disodium phosphate esters or monosodium succinate esters. Similarly, carboxyl-containing substituents at R.. or R- positions may be further modified by conversion to alkyl esters, such as methyl esters, to alkali metal salts, such as a sodium or potassium salt, or to glycosyl esters, such as a glucose or rhamnose ester. Hernandulcin is non-mutagenic when tested with Salmonella typhimurium strain TM677, and is non-toxic for mice as assessed by acute toxicity studies employing oral administration. Hernandulcin is relatively stable to hydrolysis over a wide pH range in tests conducted at temperatures up to about 60°C. Its principal decomposition products are 3-methyl- -2-cyclohexen-l-one and 6-methyl-5 hepten-2 one. These products, particularly the latter, were essen¬ tially non-toxic for mice when tested as above. These characteristics of the (racemic) (+)-hernandulcin make possible its use in a variety of beverages as a sweetening agent. Where a greater water solubility is desired a selected analog, as discussed above, may be employed. Any particular beverage formulation, containing a taste-effective concentration of the sweetening agent of this in¬ vention, may also contain other additive agents for taste modification or intensity. In general, the sweetening agents of this invention should be em¬ ployed at concentrations in the range from about 0.05 to about 0.50 grams per liter of aqueous solution. In liquid concentrates an analog having greater solubility is preferred. In solid powders, a pre¬ ferred analog is an alkali metal salt derivative inasmuch as hernandulcin and many of the specified analogs are oils. An alternative formulation employs the sweetening agent adsorbed on other solid compo¬ nents of the powered concentrate for beverage use. In any intended beverage formulation, any one or more of the sweetening agents of this invention may be employed. When employed as a sweetening agent or flavor modified in various foodstuffs, including candies and other confectioner's products, any one or combination of the agents of this invention, preferable her¬ nandulcin, may be incorporated as an additive in the desired product. The sweetening agent may be an ingredient of a recipe for sauces, dressings, or condiments. The agent may also be incorporated in canned fruits, vegetables, and the like, or in normally frozen or refrigerated products such as meats, pastries, packaged meals or entrees, or dairy products such as ice cream. When employed in a foodstuff composition the concentration of the sweetening agent of this in¬ vention should be at a taste-effective level. considering the interplay of all of the other ingre¬ dients. Generally, a concentration level within the range from about 0.005 to about 0.5 wt. % will be effective, with a preferred range being from about 0.01 to about 0.1 wt. %. The sweetening agents of this invention are particularly useful in a variety of pharmaceutical compositions as a relacement for agents such as sucrose which are often employed in high concen¬ tration. Such pharmaceutical compositions may be provided in liquid form, as thickened products, including gels, or as solid formulation, in each instance conforming generally to the concentration ranges set forth for both liquid and solid formu¬ lations. The low cariogenicity of the sweetening agents of this invention is strongly suggestive of use in various oral hygiene compositions. When employed as an ingredient in such products very low concen¬ trations can be taste-effective. A particularly preferred employment of the agents of this invention involves liquid oral hygiene compositions such as mouthwashes. Another effective use of these agents is in thickened gels or pastes employed as den¬ tifrices. Use in solid oral hygiene compositions is equally effective. Following a determination of the structure of hernandulcin, a method for synthesis of the ste- reoisomers was devised and reaction conditions were optimized to permit improvement of product yield to the range of about 50 mol. % of the theoretical value. The synthetic reaction involves the condensation of cyclic and acyclic moieties. Although the synthe¬ sis of hernandulcin is described here, the disclosed technique can be employed for synthesis of the remainder of the analogs of general formula I with only minor modifications. More specifically, hernandulcin is preferable prepared by condensing 3-methyl-2-cyclohexen-l-one with 6-methyl-5-hepten-2-one in solution in a cyclic ether solvent, such as tetrahydrofuran or dioxane, in the presence of a lithium-or boron-containing conden¬ sation agent, such as a lithium amide or a boron triflate. Suitable amides include lithium diisopropylamide, lithium cyclohexylisopropyl-amide, lithium hexamethyl-disilylamide, and lithium 2,2,6,6-tetra-methylpiperidide. A typical boron triflate is di-n-butylboron trifluororaethyl sul- fonate. Preferable the reaction solution also contains a small amount of 2,2'-dipyridyl. The reaction is conducted, with mixing, at a temperature within the range from about -78° to 0°C, preferably about -20°C. to about -10°C, and most preferably about -15°C. , and under an inert atmosphere, usually afforded by gaseous nitrogen. Agitation is continued for from about 5 to about 15 minutes and the reation mixture is quenched with the addition of a saline solution, such as 10% aqueous ammonium chloride. The reaction product is extracted from the aqueous mixture with, for example, diethyl ether, recovered by distillation of the ether solvent, and the re¬ covered product oil is distributed on high-surface area silica gel. Elution of the product, employing, for example, a hexane-acetone elution solvent, preferable comprising about 97 vol. % hexane and about 3 vol.% acetone, occurs with the hernandulcin product being recovered from selected adjacent fractions in very high purity. The following specific examples detailing the synthesis of the preferred compounds according to the present invention are offered by way of further illustration of the present invention. Although limited to the preferred compound, the examples may readily be modified by those skilled in the . art to incorporate those substituted compounds wherein "R" groups are other than the illustrated. Accordingly, the following examples are not meant to be limiting of the present invention. EXAMPLE I Dried and chopped L^ dulcis herb (900 g.) was extracted with petroleum ether (b.p. range 60-80°, 4 x 4 liters) . Extracts were combined and evaporated to produce 7.35 g. of a sweet-tasting residue. A portion (1.4 g.) of this residue was chro atographed over silica gel (particle size 0.063-0.1) mm., 120 g.) contained in a glass column (25 cm. x 1.5 cm.). Separation was achieved by gradient elution with mixtures of hexane and acetone, ranging in polarity from 100% hexane to 90% hexane-10% acetone. Frac¬ tions (10 ml. each) were monitored by thin-layer chromatography on silica gel, using hexane-acetone (9:1) as developing solvent and 60% sulfuric acid as visualizing reagent. The sweet constituent of L. dulcis was found to be present in fractions 47-59 that were eluted with 90% hexane-10% acetone. These fractions were combined, and the sweet compound was purified by preparative thin-layer chromatography on silica gel plates developed with the solvent system hexane-acetone (17:3) . Further quantities of sweet isolate were generated by repetition of the chromatographic steps outlined above on additional quantities of plant material. The sweet isolate obtained in Example I, which was named hernandulcin, was a colorless oil, [ ] 25=109° c. 0.11, ethanol) , and exhibited the molecular formula c i5 H 24°2' based on a high- resolution electron impact mass spectral measurement of the molecular ion appearing at 236.18005 amu. Infrared and ultraviolet absorption maxima typical of an alpha-beta unsaturated ketone functionality and an intramolecularly-bonded hydroxy group were observed. The H-NMR (nuclear magnetic resonance) spectum revealed the presence in hernandulcin of a tertiary methyl group on a carbon bearing the hydroxy group, and three methyl groups attached to double bonds and two olefinic protons. Based on these spectral features, hernandulcin was assigned as a monocyclic sesquiterpene of the bisabolane type. The 13HC-NMR spectrum showed discrete signals for all fifteen carbons when fully proton-decoupled, and was compar¬ able with analogous date reported for other sesquiterpenes in this series. The spectra are summarized in Table I. Table I 1H- and 13 C-NMR Spectral Data for Hernandulcin, recorded using Deuteriochloroform as Solvent with Tetramethylsilane as Internal Standard. Abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad; J, coupling constants in hertz; £ . chemical shift; M, multiplicity. Chemical shifts were assigned by selective double-irradiation experiments. 3-H-NMR* 13 C-NMRf Carbon <_ * ,M,J.Hz) A, M C-l 204.0,s C-2 5.88,s(br) 127.4,d C-3 163.6,s C-4 2.34 ,m 31.2,t C-5 1.69 ,m;2.05,m 25.0,t C-6 2.42,dd,14.1,4.5 52.0,d C-7 1.97,s(br) 24.1,q C-l' 73.9,s C-2' 1.48,t(br) ,8.0 40.1,t C-3 ' 2.05,m;2.15,m 21.5,t C-4' 5.19,t(br),8.1 124.4,d C-5* 131.4,s C-6' 1.68,s(br) 25.7,q C-7' 1.63,s(br) 17.6,q C-3' 1.18,s 23.6,q OH 5.29 ,s *Recorded at 360 MHz. fRecorded at 90.8 MHz. Additional evidence for the structure of this compound was obtained by the identification of two principal degradation products, 6-methyl-5-hepten- 2-one and 3-methyl-2-cyclohexen-l-one, produced when hernandulcin was treated with strong bases or heated to 140°C. These products were spectrally identical to authentic samples obtained from commercial sources, and consistent with the tendency of other beta-ketols to experience reverse-aldol condensation when similarly treated. The relative configuration of hernandulcin was inferred not only by the existence of strong hydrogen bonding between the hydroxy and carbonyl groups, but also because of the demonstrated absence in its H-R spectrum of any nuclear Overhauser enhancement. The latter would be expected by the interaction of the C-14 methyl protons and the C-6 proton, if, in fact, these functionalities were arranged in a syn confor¬ mation. Thus, the number of possible structures for natural hernandulcin was reduced to two, namely, either (6R,'R)- or (6S,2"S_)-6- (1' ,5'-dimethy1-1- *hydroxyhex-4'-en-l-yl)-3-methyl-2-cyclohexen-l-one. i I ) EXAMPLE II Optimized reaction conditions were employed in the synthesis of hernandulcin. To a cold (-15°C.) solution of lithium diisopropyland.de (0.92 g. ; 8.5 mmol.) and a few mg. of 2,2*-dipyridyl in 15 ml. anhydrous tetrahydrofuran, 0.93 g. (8.5 mmol.) of 3-methyl-2-cyclohexen-l-one were added. The reaction mixture was maintained under an atmosphere of nitro¬ gen, and then 1.07 g. (8.5 mmol.) of 6-methyl-5- -hepten-2-one were incorporated. After stirring for 5 in. , the reaction was quenched with 10% aqueous ammonium chloride, and the product extracted into diethyl ether (3 x 20 ml.). The combined diethyl ether fractions were washed with 15 ml. of a satura¬ ted solution of sodium chloride, and were dried over anhydrous sodium sulfate. On removal of solvent, 1.9 g. of a yellow liquid were obtained. Purification over silica gel (0.05-0.2 mm.; 250g.) by elution with exane-acetone (97:3) produced pure hernandulcin (970 mg. ; 4.1 mmol.). The synthetic hernandulcin was sweet, .and was indistinguishable from natural hernandulcin in its spectroscopic (UV, IR, NMR, MS) and chromatographic (TLC) characteristics. EXAMPLE III The acute toxicity of synthetic hernandulcin, 3-methyl-2-cyclohexen-l-one, and 6-methyl-5- hepten-2-one was determined in male Swiss-Webster mice. Compounds were suspended in a minimum volume of 1% sodium carboxymethylcellulose, and were admin¬ istered by oral intubation at the doses indicated in Table II. Mortality was determined up to 14 days after compound administration, and it may be seen from Table II that only 3-methyl-2-cyclohexen-l-one exhibited an D 5Q of less than 2 g/kg, as expressed after 24 hours. Hernandulcin and 6-methyl-5-hepten-2-one produced no mortality at doses up to 1 g/kg body weight. In Table III, body weight variations are shown over a two-week period for synthetic hernandulcin and 6-methyl-5-hepten-2-one. For hernandulcin there were no significant variations from control values for the body weights in mice dosed at 1.0 g/kg and 2.0 g/kg. In corresponding studies performed on 6-methyl- -5-hepten-2-one, however, the Day 14 total body weight values were significantly different from control values. Table II Acute Toxicity Testing of Hernandulcin, and the Hernandulcin Decomposition Products 3-Methyl-2-cyclohexen-l-one and 6-Methyl-5-hepten-2-one, Following Oral Administration fo Mice a Compound Dose Administered (g/kg) Estimated LD50 (g/kg) 1.0 2.0 Hernandulcin b ' c 0/10 d 0/10 d >2.0 3-Methyl-2-cyclo- b hexen-1-one 8/10 e ' f 1.6 + 0.13 6-Methyl-5-hepten- b 2-one 0/9 d 0/9 d >2.0 a Male Swiss-Webster mice (9 or 10 animals per dose). b Purity > 99%. c Synthetic compound used [mixture of two enantiomers, i.e., (R,R)- and (S_,S_)-forms] . ^Mortality = number of mice dying within 14 days/number of starting mice. e Mortality = number of mice dying within 24 hours/number of starting mice. f Other mortality figures were: 0/10, 2/10, 4/10, 5/10 and 10/10 at, respectively, 1.1, 1.3, 1.4, 1.6, and 2.4 g/kg. Table III Variation in Body Weight in Mice Following Oral Administration of Hernandulcin and the Hernandulcin Decomposition product, 6-Methyl-5-hepten-2-one Compound Dose Body Weight (g) of Test Animals at Time Intervals 3 ' (g/kg) Pre-Drug Day 1 Day 3 Day 7 Day 14 Hernandulcin 0 ' d 1.0 e 29.3+0.8 27.1+0.8 26.7+0.8 27.9+0.8 28.1+0.8 Controls e 29.3+0.8 27.1+0.8 26.8+0.8 27.4+0.8 28.8+0.8 Kernandulcin c » d 2.0 e 26.0+0.9 25.6+0.9 26.3+0.9 26.0+0.9 27.6+0.9 Controls e 27.8+0.9 27.4+0.9 27.5+0.9 27.0+0.9 29.1+0.9 6-Methyl-5-hep- c ten-2-one l.of 27.7+0.8 28.2+0.8 26.3+0.8 24.7+0.9 28.0+0.8 6-Methyl-5-hep- c ten-2-one 2.0 f 28.8+0.8 28.6+0.8 26.8+0.9 25.0+0.9 24.5+0.89 Controls f 28.9+0.8 29.6+0.8 29.3+0.8 26.2+0.8 29.2+0.8 a No significant differences (p<0.05) were found in treated animals compared with control anima on one way analysis of variance, except where indicated. °Male Swiss-Webster mice. e Number of animals in each group = 10. c Purity > 99%. ^Number of animals in each group = 9. , d Synt-hetic compound . 9signi f ican t di f ference f rom controls on one-way anal of variance EXAMPLE IV Magnitude estimation studies of sweetness intensity and sweetness pleasantness on natural hernandulcin were performed by a human taste panel, made up of 12 volunteers who had passed a screening test administered earlier, and who were thus con¬ sidered reliable subjects. Seven concentration of sucrose (0.031, 0.062, 0.125, 0.25, 0.5, 1.0 and 2.0 M) and of natural hernandulcin (1.06 x 10 —5 4,24 x 10 "5 , 8.47 x 10 "5 , 1.69 x lθ ""4 , 3.39 x lθ "4 and 6.78 x 10 —4 M) were prepared in distilled water. So¬ lutions were presented to the subjects in a ran¬ domized fashion and were sampled in duplicate. After tasting, each solution was expectorated, and the mouth was washed with distilled water. Results of the comparison are presented in Table IV. Using an open-ended magnitude ratio scale, sucrose was rated for its sweetness intensity only, while hernandulcin was rated for sweetness, af¬ ter-taste, off-taste intensities and pleasantnesses, as well as bitterness intensity. The results, shown in Table V, indicate that the concentration of the hernandulcin solution approxi¬ mately equal to a standard 0.27 M sucrose solution in sweetness intensity was 2.5 x 10 -4 M. Thus on a molar basis, herenandulcin was perceived by the panel to be about 1,000 times sweeter than a 0.27 M sucrose solution, or about 1,500 times sweeter on a weight basis. However, hernandulcin was judged by the panel to be less pleasant than sucrose, and to exhibit significant off- and after-tastes, as well as a moderate bitterness. Table IV Comparison of Sweetness Intensity and Sweetness Pleasantness of Sucr as determined by a human taste panel using an open-ended magnitude r Concentration Tasted Sweetness Intensityt Sweetness P Sucrose(M) Hernandulcin Sucrosej Hernandulcin Sucrosetf H (MxlO -5 ) !T 0.031 1.06 6.6 (2.44) 9.7 (4.19) -3.2 (6.44) 0.062 2.11 7.8 (1.96) 8.8 (2.37) -1.6 (7.61) 0.125 4.24 21.9 (3.30) 8.8 (3.18) 19.2 (5.23) • 0.25 8.47 38.1 (4.43) 15.8 (4.10) 31.8 (6.34) 0.50 16.9 44.4 (4.63) 39.7 (3.52) 37.8 (5.96) 1.0 33.9 75.5 (4.73) 44.4 (5.45) 2.5 (9.57) 2.0 67.8 89.1 (2.93) 62.7 (5.59) -20.2 (12.69) The test panel was composed of seventeen previously trained voluntee • { • Intens ity attributes (means and standa rd errors ) were in the range o •Hedonic attributes (pleasantness-unpleasantness) carry a + or - sign standard errors) were normalized to span the range -100 (extremely un (extremely pleasant). A hedonic value of 0 is therefore neutral, nei unpleasant. NA solution of 0.27 M was determined graphically as providing a stand sucrose intensity. Table V Sensory Test Values for the Lippia dulcis Sweet Constituent, at a Concentration Which Matches the Sweetness of 0.27 M S Sweetener Concentration Sweetness Off Taste (M) Inten¬ Pleasant¬ Inten- Pleasant- I sity ness sity ness s Sucrose 0.27 40 Hernandulcinc,d 2.5 x 10- 4 40 15 33 -5 L)ata obtained by human taste panel using an open-ended magnitude scale. b ' " Data obtained by 17 participants. * Two replicates at each concentration were tested. Naturally occurring substance. In addition to the data collected and evaluated from the previous examples, data from a large number of other testing was collected and evaluated. Stability studies on synthetically made hernandulcin were carried out, with and without buffer solutions. The first of these manipulations was conducted by storage of this compound in cit- rate-phosphate-borate buffer solutions, at the pH levels 2, 3, 4, 6, 7, 8, 10, and 12. Hernandulcin was stored in each solution at a concentration of about 0.2 wt. %, at both room temperature and 60°C. , for two weeks. Solutions were exposed to the atmo¬ sphere after their constitution. Compound decomposition was assessed by counting the number of zones apparent by TLC analysis 1 hour, 3 hours, 6 hours, 24 hours, 1 week and 2 weeks after solution composition. Hernandulcin exhibited a single purple zone on silica gel plates when deve¬ loped in the solvent system hexane-acetone (17:3), and visualized with 60% sulfuric acid by heating at 110° for 5 minutes. At room temperature, which was used to indicate the stability of hernandulcin under normal handling, no compound breakdown was evident for any of the pH levels used after up to two weeks of storage. Similarly, no decomposition was observed for any solution when stored for up to 24 hours at 60°C. , a temperature chosen to represent that which might be experienced during food processing. Howev¬ er, after 1 week and 2 weeks at 60°C, substantial breakdown was apparent in all solutions, with greater degrees of decomposition being observed at the acid and alkaline pH extremes, although hernandulcin was detectable at all pH's at these time intervals. No attempt was made to quantify the degree of breakdown of hernandulcin. An assessment of the thermostability of her¬ nandulcin was conducted by storing a sample at 100°C. for 24 hours, and comparing it by TLC with the unheated compound. After such treatment, approxi¬ mately 60% of the compound remained unchanged, and about a 10% conversion to each of 3-methyl-2- -cyclohexen-1-one and 6-methyl-5-hepten- 1-one was observed. These figures represent approximations obtained by calculating TLC zone areas. Cariogenicity tests were conducted with synthe¬ tic, racemic hernandulcin. In a first test, the ability of microbes to utilize this sweetener source as a nutrient and convert it to acidic by-products was determined. S_. mutans utilized this sweetener as a carbohydrate source and produced 2.6 x 10 -5 mEq acid/min. when the concentration of the sweetener was 5,000 micrograms per unit volume. This was a rela¬ tively low rate of acid production, as compared to sucrose. Acid production from sucrose (500 micro- grams per unit volume) was 2.8 x 10 —4 mEq acid/min. under identical conditions. On a weight basis the rate of acid production was one hundred-fold less than that attained from sucrose. In a second test, the ability of the test compound to inhibit acid production from sucrose by S_. mutans was determined. The test compound failed to reduce the rate of acid production from S_. mutans, even when the test compound was used in a 10-fold concentration over sucrose (428 micrograms per unit volume sucrose and 5,000 micrograms per unit volume sweetener) . The mutagenic potential of synthetic herna¬ ndulcin was examined by a forward mutation assay utilizing Salmonella Typhimurium strain TM677, according to known procedures. No significant mutagenic activity was noted when hernandulcin was assayed either in the presence or absence of a metabolic activating system derived from the livers of Aroclor 1254-pretreated rats. Thus while we have illustrated and described the preferred embodiment of our invention, it is to be understood that this invention is capable of varia¬ tion and modification, and we therefore do not wish to be limited to the precise terms set forth, but desire to avail ourselves of such changes and alter¬ nations which may be made for adapting the invention to various usages and conditions. Accordingly, such changes and alterations are properly intended to be within the full range of equivalents, ad therefore within the purview, of the following claims. Having thus described our invention and the manner and process of making and using it, in such full, clear, concise, and exact terms so as to enable any person skilled in the art to which it pertains or with which it is most nearly connected to make and use the same;

Claims: WE CLAIM: 1. 6- ( l * , 5 ' -dimethyl-l ' -hydroxyhex- -4 ' -en-l ' -yl) -3-methyl-2-cyclohexen-l-one . 2. 6-Hydroxyalkyl derivatives of about 8; R- is an ethyl, propyl, butyl, methylol or carboxymethyl group; R~ is hydrogen or an ethyl, propyl, or butyl group; and R 3 is a methyl, ethyl, propyl, butyl, allyl, methallyl, methylol, hydr¬ oxyethyl, or carboxyethyl group. 3. The 6-hydroxyalkyl derivates of claim 2 wherein the R- substituent additionally includes a methylol group modified to comprise a disodium phosphate ester or a monosodium succinate ester thereof. 4. The 6-hydroxyalkyl derivates of claim 2 wherein the R_ substituent additionally includes a methylol or hydroxyethyl group modified to comprise a disodium phosphate ester or a monosodium succinate ester thereof. 5. The 6-hydroxyalkyl derivates of Claim 2 wherein the R.. substituent additionally includes a carboxymethyl group modified to comprise a methyl ester, alkali metal salt, or glycosyl ester thereof. 6. The 6-hydroxyalkyl derivatives of claim 2 wherein the R_ substituent additionally includes a carboxyethyl group modified to comprise a methyl ester, alkali metal salt, or glycosyl ester thereof. 7. Beverage compositions comprising a taste-effective concentration of a low-cariogenic, water-soluble sweetening agent selected from the class consisting of 6-(1* ,5'-dimethyl 1-hydroxyhex- -4'-en-1'-yl)-3-methyl-2-cyclohexen-l-one, -hydro- xyalkyl derivates of 2-cyclohexen-l-one, and mixtures thereof.6.- 8. The beverage compositions of claim 7 wherein the concentration of sweetening agent is within the range from about 0.05 to about 0.50 grams per liter of aqueous solution. 9. Food compositions comprising^ a taste-effective concentration of a low-cariogenic, water soluble sweetening agent selected from the class consisting of 6-(l',5' dimethyl'1 • -hydro- xyhex-4'-en-l-yl)-3-methyl-2-cyclohexen-l-one, 6-hydroxyalkyl derivatives of 2-cyclohexen-l-one, and mixtures thereof. 10. The food compositions of claim 9 wherein the concentration of sweetening gent is within the range from about 0.005 to about 0.5 wt. %. 11. Pharmaceutical compositions comprising a taste-effective concentration of a low-cariogenic, water-soluble sweetening agent selected from the class consisting of 6-(1' ,5"-dimethyl-1'-hydroxyhex-4'-en-1'-yl)-3-methy- 1-2-cyclohexen-l-one, 6-hydroxalkyl derivatives of 2-cyclohexen-l-one, and mixtures thereof. 12. Liquid pharmaceutical compositions of claim 11 wherein the concentration of sweetening agent is within the range from about 0.05 to about 0.50 grams per liter of aqueous solution. 13. Thickened oral hygiene compositions of claim 11 wherein the concentration of sweetening agent is within the range from about 0.005 to about 0.5 wt. %. 14. Solid pharmaceutical compositions of claim 11 wherein the concentration of sweetening agent is within the range from about 0.005 to about 0.5 wt. %. 15. Pharmaceutical compositions of claim 11 comprising oral hygiene compositions. 16. A process for the preparation of hernandulcin, comprising the steps of: a) affording a medium comprising a solution of a lithium- or boron-containing condensing agent in a cyclic ether solvent; b) mixing and agitating equimolar quan¬ tities of 3-methyl-2-cyclohexen-l-one and 6-methyl- -5-hepten-l-one in said medium, to effect conden¬ sation thereof; c) quenching the reaction mixture by addition of anaqueous saline solution and extracting the condensation product therefrom; d) separating the condensation product from the extract and distributing the separated material upon high-surface area silica gel; e) fractionating the silica-gel dis¬ tributed material by elution into a series of frac¬ tions with a hexane-acetone solution; and f) recovering substantially pure racemic hernandulcin from selected fractions of eluate. 17. The process of claim 16 wherein the tetrahydrofuran additionally contains a trace amount of 2,2'-dipyridyl. 18. The process of claim 16 wherein the lithium-containing condensing agent is selected from the class consisting of lithium diisopropylamide, lithium cyclohexylisopropylamide, lithium hexameth- yldisilylamide, and lithium 2,2,6,6-tetramethyl- -piperidide. 19. The process of claim 16 wherein the boron-containing condensing agent is a boron triflate. 20. The process of claim 16 wherein the cyclic ether solvent is tetrahydrofuran or dioxane. 21. The process of claim 16 wherein the saline solution is an aqueous ammonium chloride solution. 22. The process of claim 16 wherein the hexane-acetone solvent comprises about 97 vol. % hexane and about 3 vol. % acetone. 23. The process of claim 16 wherein the condensation reaction is conducted in an inert atmosphere. 24. The process of claim 16 wherein the condensation reaction is effected at a temperature within the range from about -78° to about -0°C. 25. The process of claim 24 wherein the reaction temperature is within the range from about -20° to about -10°C. 26. The process of claim 16 wherein the condensation reaction is effected by agitating the mixture for a time within the range from about 5 to about 15 minutes.

Inventors: COMPADRE CESAR M, KINGHORN ALAN DOUGLAS, PEZZUTO JOHN M, COMPADRE, CESAR, M., KINGHORN, ALAN, DOUGLAS, PEZZUTO, JOHN, M.
Applicants: UNIV ILLINOIS, UNIVERSITY OF ILLINOIS
Application year: 1985
Main classification: A61K7
IPC codes: A23L1, C07C45, C07C49, C07C51, C07C57, C07C59, C07C67, C07F9