ERGOSTADIENTRIOLS,COMPOSITIONS CONTAINING SAME,AND METHODS OF PREPARING AND USING SAME

Abstract: Ergostadientriols which may be recovered from either the fungus R. oligosporus or the fungus R. oryzae as well as from soybeans which have been fermented with either of these fungi. Alternatively, the sterols may be chemically synthesized. The sterols of this invention are antioxidants which may be used in antioxidant compositions alone or preferably with one or more isoflavone. Of particular significance is the discovery that the ergostadientriols of this invention are useful in lowering serum cholesterol levels. Accordingly, this invention in a particularly preferred embodiment discloses pharmaceutical compositions containing one or more ergostadientriol. Finally, therapeutic methods which utilize pharmaceutical compositions in accordance with this invention are disclosed.

Description: ERGOSTADIENTRIOLS, COMPOSITIONS CONTAINING SAME, AND METHODS OF PREPARING AND USING SAKE BACKGROUND OF THE INVENTION This is a continuation-in-part of Application Serial No. 804,594, filed June 8, 1977, the disclosure of which is hereby incorporated by reference into the present disclos¬ ure. It has heretofore been known that antioxidant properties are possessed by tempeh, a fermented soybean product ob¬ tained by fermenting soybeans with a fungus, either Rhizo- ous oligosOorus or Rhizoous orvzae. Food products contain- ing te peh, such as fish or fatty meat food products exhi¬ bit improved stability, see U.S. Patent 3,681,085 (1972). Further, it has heretofore been found that by extracting tempeh with a mixture of hexane and ethanol, a component of tempeh, namely oil of tempeh, can be recovered, see U.S. Patents 3,762,933 (1973) and 3,855,256 (1974), which exhibits enhanced antioxidant properties relative to those of tempeh. It has also been previously known that the av¬ erage serum cholesterol levels of persons living in Indo¬ nesia where tempeh is a food staple are reduced relative to those of persons living in Western countries. SUMMARY OF THE INVENTION The present invention involves the discovery that ergostadi¬ entriols which, possess antioxidant properties are produced by the fungi Rhizopus oligosporus and Rhizopus oryzae and may be recovered therefrom. Alternatively, these ergosta¬ dientriols may be chemically synthesized. The ergostadien¬ triols of this invention have the structure: OMPI wherein X may be hydrogen or a hydroxyl group provided that X twice be hydroxyl. This structure is intended to cover both o and S stereoisomers of the substituents at the 5 and 7 positions and accordingly for the purposes of this applica¬ tion no distinction shall be made between the α and 8 isomers These compounds possess antioxidant properties and may be ut¬ ilized in the stabilization of a wide variety of food prod¬ ucts including edible fats and oils. Furthermore, antioxi- dant compositions which include one or more ergostadientriol in accordance with the present invention and one or more iso- flavone have been found to be espeically effective in stabil¬ izing various food products. A particularly significant aspect of the present invention in volves the discovery that pharmaceutical compositions which include one or more ergostadientriol having the structure set forth hereinabove and a pharmaceutically acceptable carrier are useful in reducing serum cholesterol levels. One or more of the ergostadientriols of the present invention may be prepared by culturing the fungus R^ oligosporus or the fungus R^ oryzae and recovering the compound or compounds therefro . The fungus typically produces a mixture of ergo¬ stadientriols in the presence of ultraviolet light. Speci¬ fic sterols can then be separately recovered. If the fungus is cultured in the present of visible light, the formation of one of the sterols is favored, specifically, the ergosta¬ dientriol having the structure: Compound II can then be isolated and recovered from the fun¬ gus. Additionally, one or more of the ergostadientriols can be prepared by fermenting soybeans with. R^_ oligosporus or R. oryzae, contacting the resulting fermented soybean product, i.e. tempeh, with methanol to separate from the fermented soy¬ bean product an extract containing one or more of the ergo¬ stadientriols and recovering the compound or compounds from the methanol extract. As is the case where the ergostadien¬ triols are prepared by culturing of one of the fungi identi¬ fied hereinabove, the presence of visible light during fermen¬ tation of the soybeans results in preferential formation of ergostadientriol II. Alternatively, the ergostadientriols may be prepared by chemical synthesis. Finally, the present invention provides methods of preventing and/or treating diseases involving increased serum ' cholester¬ ol levels which involve administering an effective amount of a pharmaceutical composition such as described herein to a pa ient or subject having an abnormally high serum cholesterol lev Thus, it is a primary object of the present invention to pro¬ vide pharmaceutical compositions which include one or more er gostadientriol and a pharmaceutically acceptable carrier. It is another object to provide a novel compound useful in pharmaceutical and/or antioxidant compositions. It is a related object to provide methods of preparing such compounds. It is still another, object to provide methods of preventing and/or treating diseases which are associated with, increased serum cholesterol levels. Such therapeutic methods involve administration of an effective amount of a pharmaceutical composition which includes one or more of the ergostadientri- ols of the present invention. It is a final object of this invention to provide antioxidant compositions which include one or more of the ergostadientri¬ ols in accordance with this invention. How these and other objects of this invention are accomplishe< will become apparent upon reading the detailed description of the invention including the examples set forth, and the claims which follow. In at least one embodiment of the present in- vention at least one of the foregoing objects will be achievec DETAILED DESCRIPTION OF THE INVENTION In accordance with one embodiment of this invention, ergosta¬ dientriols having the structure: wherein X may be hydrogen or a hydroxyl group provided that X twice be hydroxyl have been prepared. These novel ergostadi¬ entriols possess antioxidative properties. They are "Ξmmerie Engel" positive at the same order of magnitude as Vitamin Ξ, that is, they reduce Fe' to Fe + ' + at room temperature, the latter forming a brilliant red complex in the presence of α, α-dipyridil. In combination with various isoflavones, these sterols provide antioxidant compositions with exceptional prop¬ erties. The ergostadientriols I have been characterized by ultraviolet, infrared, and high resolution mass spectrometry as 3-hydroxy-ergosterols. The molecular formula of these com¬ pounds is C 2 8 H 46°3' an< ^ t *^ e *^ r molecular weight 430. One such ergostadientriol has been identified as having the structure: /02027 -6- This ergostadientriol is a known compound, e.g., see Fieser and Fieser, Steroids, p.98, (compound identified as Triol 1(6 Reinhold Publishing Corporation, New York (1959) . However, i usefulness in reducing serum cholesterol levels has not previ ously been taught or suggested. Another ergostadientriol has been identified as having the fo. lowing structure: This ergostadientriol is a novel compound. As indicated hereinabove, in a preferred embodiment, the pres¬ ent invention provides pharmaceutical compositions which are useful in reducing serum cholesterol levels. Such pharmaceu¬ tical compositions comprise one or more compound having the wherein X may be hydrogen or a hydroxyl group provided that X twice be hydroxyl, and a pharmaceutically acceptable carrier. Particularly preferred for use in such pharmaceutical compo?' - tions in ergostadientriol II which has the hydroxyl groups at the 5 and 8 positions. As used herein, pharmaceutically ac¬ ceptable carrier includes without limitation all of the conven¬ tional carriers. Merely by way of illustration the following are set forth, namely, talc, starch, lactose, tragacanth, and magnesium sterate. Countless others are useful in the prac¬ tices of the present invention and the choice of any specific carrier is one well within the skill of a person in the art. Such pharmaceutical compositions may include one or more of the ergostadientriols of the present invention in amounts by weight ranging from about 0.001 to 10 percent,perferably, about 0.01 to 1.0 percent based upon the weight of said compositions. By administering such compositions to a patient or subject in an effective amount, such as an amount in the range 0.01 - 10 g per kg of the patient's or subject's weight, serum cholesterol levels can be reduced. Accordingly, the present invention provides methods of preventing and/or treating diseases asso¬ ciated with increased serum cholesterol levels, such as ather¬ osclerosis, which comprises administering to a subject or pat- ient an effective amount of a pharmaceutical composition in accordance with the present invention. In accordance ' with another emobidment of this invention, an¬ tioxidant compositions may be prepared which include one or more of the ergostadientriols I. As is disclosed more fully in co-pending, co-assigned application Serial No. 804,594, filed June 8, 1977 referred to hereinabove and incorporated by reference into the present disclosure, compositions con¬ taining one or more ergostadientriol and one or more isofla- vone provide exceptionally effective antioxidative properties. Specifically, antioxidant compositions which include an ergo¬ stadientriol having the structure: wherein X may be hydrogen or a hydroxyl group provided that X twice be hydroxyl, and one or more isoflavone having the stru ture: wherein the dashed lines may be carbon-carbon single bonds or carbon-carbon double bonds, and wherein X may be two hydrogen atoms or oxygen, and further wherein each of R, R' and R" may be a methyl group or hydrogen provide exceptional anti¬ oxidative properties as set forth in the examples herein- below. Such antioxidant compositions can be included in food prod¬ ucts to produce stabilized food compositions. Accordingly, food products such as fish, fatty meat or derivatives there¬ of, may be stabilized by the addition thereto of an antiox- idant composition which includes one or more of the ergosta¬ dientriols described hereinabove in an amount from about 0.001 to 10 per cent by weight. Additionally, stabilized edible oil and/or fat compositions may be prepared by includ¬ ing in edible oils or fats an antioxidant composition which includes one or more of the ergostadientriols of the present invention. Effective amounts of such antioxidant composi¬ tions in terms of improving the stability of oils or fats, such as for example, lard, corn oil, olive oil, soybean oil or palm oil, and the like are amounts in the range 0.01 to 1.0 per cent by weight, more or less. The ergostadientriols I may be produced by fermentation of soybeans with a fungus, either L_ oligosporus or R^ oryzae. The ergostadientriols may also be produced by and recovered from either of these fungi directly after growth on a suit¬ able culture medium. Suitable fungi for producing the ergo¬ stadientriols are Rhizopus oligosporus ATCC No. 22959 and Rhizopus oryzae ATCC No. 9363. If the fermentation of soy¬ beans with fungus , or the culturing of fungus directly is carried out in the presence of ultraviolet light, a mixture of ergostadientriols, each of which falls within the scope of structure I is produced including compounds II and III. How¬ ever, it has been found that if the fermentation or culturing is carried out in the presence of visible light, there is a preferential formation of ergostadientriol II which has the hydroxyl groups at the 5 and 8 positions as shown hereinabove. The ergostadientriol or ergostadientriols so prepared may be recovered in the following manner. Dry, e.g., lyophilized, tempeh powder or cultured fungus is contacted with a 60-70% aqueous methanol solution for. an ex¬ tended period of time, for example, overnight, at a tempera- ture of about 4°C. thereby producing an extract of ethanol- soluble components including one or more of the ergostadien¬ triols I. The methanol extract solution, after removal of insoluble material, is evaporated to dryness, preferably in vacuo, at an elevated temperature, for example, about 40-60° A solid residue is produced, ost of which is redissolvec upon contact with dry methanol. That portion of the residue which is methanol insoluble is separated * from the soluble components by centrifugation and discarded. After centrifu- gation the methanol supernatant is extracted " with hexane sev eral times, for example, two to three times, in order to re¬ move any traces of hexane-soluble impurities, such as lipids After discarding the resulting hexane extract, the remaining methanol supernatant is evaporated to reduce its volume to a minimal fraction, for example, about 20 ml, and kept at a temperature of about -20°C. for about 15-20 minutes. This results in formation of additional precipitate which is re¬ moved and discarded. The ergostadientriols may then be recovered from the methano supernatant or extract as follows. The supernatant is sub¬ jected to molecular sieve chromatography, for example, chrom atography on Sephadex LH20 using a suitable size column, for example, 2 x 40 cm, and a suitable mobile phase, for example n-propanol:ethylacetate: ater in a ratio 5:5:1. One of the fractions resulting from this chromatographic separation is fluorescent with emission in the blue range of the visible spectrum. This blue fluorescent fraction is separated and subjected to adsorption chromatography on a suitable matrix, for example, silica gel, using an appropriate mobile phase, for example, ethylacetate-.propanol:water = 95:2:3. The re¬ sulting blue flurorescent fraction is then re-chromatographe on an adsorptive matrix, for example, silica gel again, em¬ ploying a different mobile phase, for example, cyclohexane: dichloromethane:ethyl formate:formic acid = 35:30:30:5. Eac of the ergostadientriols present can then be recovered. in essentially pure form using its differential mobility on the silica gel plate. Additionally, the ergostadientriols may also be obtained in pure form from the methanol supernatant or extract by prep¬ arative high pressure liquid gas chromatography. Alternatively, the ergostadientriols may be chemically syn¬ thesized. For example, ergostadientriol II may be prepared from ergosterol by the method outlined in Fieser and Fieser, Steroids, • p. 98 and referred to hereinabove. In addition to the ergostadientriols discussed hereinabove it would appear that compounds having the structure wherein R χ may be —OH, =0,—H, —0—Alkyl, —0— cyl,— COOH —COOAlkyl or C=N and wherein R, may be — —CH,,— thout limitation th various sterochemically possible connections of Rings A,3,C an D, the reduction and addition reaction products which may be obtained by reactions involving the double bond on C g , parti¬ cularly the epoxidation and hydroxylation products, and com¬ pounds having an additional double bond between C g and C, , ma be useful in lowering serum cholesterol levels. Example I Ergostadientriol II was compared with β-sitosterol, a commer¬ cially available hypocholesterolemic agent to determine its e feet upon serum cholesterol levels in chicks feed a high chol esterol diet. In the bioassey there were four groups of three pens per grou; and five chicks per pen. Each group was fed a high cholester diet for seven days prior to blood sampling. One group serve> as control. Two groups were fed β-sitosterol at the level of 1.0% and 0.1% of the feed and one group was fed ergostadientr ol II at a level of 0.1% of the feed. The results were as fo lows: Serum Cholesterol (mg%) Control 330 . 7 * 15 . 8 0.1% β-sitosterol 333 .3 ± 7 . 9 1.0% S-sitosterol 286. 9 * 14 . 9 0.1% ergostadientriol II 283 . 8 i 23 . 4 ^ ? - ^ Statistical analysis of the data shows that the addition of 0.1% ergostadientriol II to the feed decreased serum choles¬ terol by 14.2% compared to the controls. 1.0% S-sitostero., was required to reduce cholesterol by 13.2% compared to the controls. Thus, ergostadientriol II was more than 10 times as active as β-sitostarol in reducing serum cholesterol. The results of this test would indicate to one skilled in the art that the ergostadientriol would be effective in reducing serum cholesterol levels in human patients. Further¬ more, these results would strongly suggest to one skilled in the art that the ergostadientriol would be useful in treating and/or preventing diseases associated with increased serum cholesterol levels such as athersclerosis. Exam -*-le II In a standard test assay involving oxidation of lard by ex- posure to air at 60°C. for 72 hours, ergostadientriol IH may be added at a concentration of 0.1 per cent by weight. This ad¬ dition of the ergostadientriol should result in about 50% protec¬ tion against oxidation. Example III The same test as in Example II may be carried out except that the concentration of ergostadientriol III is 0.01 per cent by weight. This reduced concentration should still provide about 25% protection against oxidation. Example IV An antioxidant composition may be prepared by mixing ergo- stadientriol III and an isoflavone having the structure: When added to lard at concentrations of each component of 0.01%, this composition should provide essentially 100% protection against oxidation in the standard assay. Example V An antioxidant composition may be prepared by mixing ergo¬ stadientriol II and an isoflavone having the structure At a concentration of 0.01-0.1% by weight of each compound this antioxidant composition would provide substantial pro¬ tection against oxidation. As will be obvious to one skilled in the art, many modifica¬ tions, variations, alterations and the like may be made in the practices of this invention without departing from the spirit and scope thereof as set forth in the claims which follow. -£\J EA"£ O P

Claims: AMENDED CLAIMS (received by the International Bureau on 7 May 1980 (07.05.80)) 1. A composition useful as an antio-xidant which, comprises an effective antioxidant amount of a compound having the wherein X may be hydrogen or a hydroxyl group provided that X twice be hydroxyl and a carrier selected from the group consis ting of edible foods , fats and oils . 2. A composition in accordance with Claim 1 which comprises the compound having the structure: - U REΛ OMPI 3. A composition in accordance with. Claim 1 which comprises a compound having the structure: 4. A composition in accordance with Claim 1 which additional¬ ly comprises one or more isoflavone having the structure: wherein the dashed lines may be carbon-carbon single bonds or carbon-carbon double honds , and wherein X may be two hydrogen atoms or oxygen, and further wherein each of R, R' and R" may be a methyl group or hydrogen . - JR 5. A pharmaceutical composition useful in reducing serum cholesterol levels which comprises an effective hypo¬ cholesterolemic amount of a compound having the structure: wherein X may be hydrogen or a hydroxyl group provided that X twice be hydroxyl and a conventional pharmaceutically acceptable carrier. Q 6. A pharmaceutical composition in accordance with Claim 5 which comprises the compound having the structure: 7. A pharmaceutical composition in accordance with Claim 5 which comprises the compound having the structure: 8. A method for preparing one or more compounds having the wherein X may be hydrogen or a hydroxyl group provided that X twice be hydroxyl which comprises culturing the fungus R^ oli¬ gosporus or the fungus R^ oryzae and recovering said compound or compounds therefrom. 9. A method in accordance with Claim 8 for preparing a compound having the structure: which comprises culturing the fungus R^_ oligosporus or the fungus R^ oryzae and recovering said compound therefrom. 10. A method in accordance with Claim 8 for preparing a compound having the structure: which comprises culturing the fungus R^ oligosporus or the fungus R_ ; _ oryzae in the presence of visible light and recov¬ ering said compound therefrom. -1TU £A> 11. A method for preparing one or more compounds having the structure: wherein X may be hydrogen or a hydroxyl group provided that X twice by hydroxyl which comprises fermenting soybeans, con¬ tacting the resulting fermented soybean product with methanol to separate from said fermented soybean product an extract containing said compound or compounds and recovering said compound or compounds from said methanol extract. 12. A method in accordance with Claim 11 wherein said fer¬ mented soybean product is tempeh. 13. A method in accordance with Claim 11 wherein said extract containing said compound or compounds is obtained by contact¬ ing said fermented soybean product with an aqueous methanol solution at a temperature in the range about 0-10°C, evapor¬ ating the resulting methanol extract solution to dryness to produce a residue, redissolving a portion of said residue in dry methanol, separating and discarding the methanol insoluble portion of said residue, contacting said dry methanol solutior. containing said redissolved residue ' with hexane to remove con¬ taminating material, discarding the resulting hexane extract, reducing the volume of- the remaining methanol extract by evap¬ oration, keeping said reduced methanol extract at a reduced temperature for 10-30 minutes so as to produce a precipitate removing and discarding said precipitate, and recovering saic compound or compounds from the remaining methanol extract. 14. A method in accordance with Claim 11 wherein said recov¬ ery of said compound or compounds from said methanol extract comprises chromatographic separation, said chromatographic separation being molecular sieve chromatography followed by adsorption chromatography. 15. A method in accordance with Claim 11 for preparing a com¬ which comprises fermenting soybeans, contacting the resulting fermented soybean product with methanol to separate from said fermented soybean product an extract containing said compound, and recovering said compound from said methanol extract. 16. A method in accordance with Claim 11 for. preparing a compound having the structure: - υSEΛ» /02027 - 32 - which comprises fermenting soybeans in the presence of visi¬ ble light, contacting the resulting fermented soybean product with methanol to separate from said fermented soybean product an extract containing said compound, and recovering said com¬ pound from said methanol extract. 17. A method of preventing and/or treating diseases involv¬ ing increased serum cholesterol levels which comprises admin¬ istering to a subject an effective amount of a pharmaceutical composition in accordance with Claim 5. 18. A method of preventing and/or treating diseases involv¬ ing increased serum cholesterol levels which comprises admin¬ istering to a subject an effective amount of a pharmaceutical composition in accordance with Claim 6. 19. A method of preventing and/or treating diseases involv¬ ing increased serum cholesterol levels which comprises admin¬ istering to a subject an effective amount of a pharmaceutical composition in accordance with Claim 7. 20. A method in accordance with Claim 17 wherein said dis¬ ease to be treated is atherosclerosis. 21 . A method in accordance with claim 17 wherein said effective amount of pharmaceutical composition is an amount in the range 0 .0.1 - 10 mg per kg of the subject ' s weight. 22. A compound having the structure 23. A pharmaceutical composition useful in reducing serum cholesterol levels which comprises an effective hypo¬ cholesterolemic amount of a compound having the structure: wherein R, may be — OH , =0 , — H , — O —Alky 1 , - 0 * — Acyl , — COOH , — COOAlkyl or CΞN and wherein R- may be —H and a pharmaceutically accept¬ able carrier. 24, A method of preventing and/or treating diseases involvi increased serum cholesterol levels which comprises administe ing to a subject an effective amount of a pharmaceutical com position in accordance with Claim 23. 25. A method in accordance with Claim 24 wherein said effec tive amount of pharmaceutical composition is an amount in th range 0.01 - 10 mg per kg of the subject's weight. 26. A method of preventing and/or. treating h per- chol s-terolem a which- comprises administering to a subject an effective amount of a .pharmaceutical composition in accordance, with, any of Claims- 5, 6, 7 or 23-

Inventors: ZILLIKEN F
Applicants: Z L PARTNERSHIP LTD
Application year: 1979
Main classification: C07J9
IPC codes: A23L3, A61K31, C07J1, C07J9, C09K15, C11B5, C12P33