阅读说明：本技术 降低ldl-胆固醇的细胞提取物和食品补充剂 (Cellular extracts and food supplements for lowering LDL-cholesterol ) 是由 鲁迪·瓦提兹 菲利斯·马斯特罗里奥 罗伯特斯·克里斯蒂安·约瑟夫斯·萨特斯 于 2018-06-14 设计创作，主要内容包括：本发明涉及降低低密度脂蛋白(LDL)-胆固醇的制剂,具体涉及用作用于降低血浆LDL-胆固醇水平的药物的细菌细胞提取物,其中在将所述细胞与所述混合物混合时,在8℃至37℃下提取10分钟至48小时获得所述提取物,以及其中用10:1至1:10体积比的具有60℃至80℃的沸点的石油醚和包含氯化钠的甲醇的混合物提取所述降低ldl-胆固醇的细胞提取物和食品补充剂降低ldl-胆固醇的细胞提取物和食品补充剂深红红螺菌(Rhodospirillum rubrum)细胞。更具体地,本发明涉及通过用具有60℃至80℃的沸点的石油醚和包含氯化钠的甲醇的混合物提取深红红螺菌细胞而可获得的深红红螺菌的石油醚提取物。本发明还涉及本发明的深红红螺菌的石油醚提取物,其用于降低个体血浆中的LDL-胆固醇的方法中。本发明还涉及药物制剂、含有所述制剂的食品补充剂、含有所述食品补充剂的食品以及它们的制备方法。(The present invention relates to a formulation for reducing Low Density Lipoprotein (LDL) -cholesterol, in particular to a bacterial cell extract for use as a medicament for reducing plasma LDL-cholesterol levels, wherein said extract is obtained by extraction at 8 ℃ to 37 ℃ for 10 minutes to 48 hours when mixing said cells with said mixture, and wherein said LDL-cholesterol reducing cell extract and food supplement Rhodospirillum rubrum (Rhodospirillum rubrum) cells are extracted with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and sodium chloride containing methanol in a volume ratio of 10:1 to 1: 10. More specifically, the present invention relates to a petroleum ether extract of rhodospirillum rubrum obtainable by extracting cells of rhodospirillum rubrum with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol comprising sodium chloride. The invention also relates to a petroleum ether extract of rhodospirillum rubrum of the invention for use in a method of reducing LDL-cholesterol in the plasma of an individual. The invention also relates to pharmaceutical formulations, food supplements containing said formulations, food products containing said food supplements and methods for their preparation.)

1. A petroleum ether extract of Rhodospirillum rubrum, obtainable by extracting Rhodospirillum rubrum cells with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol comprising sodium chloride, wherein the extract is obtained by extraction at 8 ℃ to 37 ℃ for 10 minutes to 48 hours when mixing the cells with the mixture, and wherein the Rhodospirillum rubrum cells are extracted with a mixture of the petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol comprising sodium chloride in a volume ratio of 10:1 to 1: 10.

2. The petroleum ether extract of rhodospirillum rubrum according to claim 1, wherein the extract is obtained by extraction at 10 ℃ to 30 ℃, preferably 12 ℃ to 27 ℃, more preferably 15 ℃ to 25 ℃, for 20 minutes to 24 hours, preferably 30 minutes to 16 hours, more preferably 45 minutes to 8 hours, most preferably 1 hour to 3 hours, when mixing the cells with the mixture.

3. The petroleum ether extract of rhodospirillum rubrum according to claim 1 or 2, wherein the extract is obtained by centrifuging the mixture of petroleum ether having a boiling point of 60 to 80 ℃ and methanol comprising sodium chloride at 15 to 25 ℃ for about 10 to 60 minutes after incubating rhodospirillum rubrum cells with the mixture.

4. The petroleum ether extract of rhodospirillum rubrum according to any of claims 1 to 3, wherein the cells of rhodospirillum rubrum are extracted with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol comprising sodium chloride in a volume ratio of 8:1 to 1:8, preferably 5:1 to 1:5, more preferably 2:1 to 1:2, most preferably 1: 1.

5. The petroleum ether extract of rhodospirillum rubrum according to any of claims 1 to 4, wherein the extract is obtained by extraction at a temperature of 15 ℃ to 25 ℃ for 1 to 3 hours when the cells are mixed with the mixture, and wherein the rhodospirillum rubrum cells are extracted with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol comprising sodium chloride in a 1:1 volume ratio.

6. The petroleum ether extract of rhodospirillum rubrum according to any one of claims 1 to 5, wherein the rhodospirillum rubrum cells are extracted with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol comprising about 0.02 to 0.04 wt% of sodium chloride, preferably about 0.03 wt% of sodium chloride.

7. The petroleum ether extract of rhodospirillum rubrum according to any one of claims 1 to 6, wherein the extract comprises one or more carotenoids and/or one or more quinones.

8. The petroleum ether extract of rhodospirillum rubrum according to claim 7, wherein the extract comprises one or more carotenoids selected from the group consisting of the carotenoids rhodol, 1-hydroxy-spiroxanthin, 3, 4-didehydropurpurin b, chloroxanthin, bacteriopheophytin a, rhodorubin b, spiroxanthin, 3, 4-dihydrospiroxanthin and the phytyl derivatives of bacteriopheophytin a, and/or one or more quinones selected from the group consisting of the following quinones: ubiquinol-10, ubiquinone-9, ubiquinone-10, and crimson quinone-10.

9. The petroleum ether extract of rhodospirillum rubrum of claim 7, wherein the extract comprises rhodol, 1-hydroxy-spiroxanthin, 3, 4-didehydropurpurin b, chloroxanthin, bacteriopheophytin a, rhodorubin b, spiroxanthin, 3, 4-dihydrospiroxanthin, a phytyl derivative of bacteriopheophytin a, ubiquinol-10, ubiquinone-9, ubiquinone-10, and rubroquinone-10.

10. The petroleum ether extract of rhodospirillum rubrum according to any of claims 1 to 9 for use as a medicament.

11. The petroleum ether extract of rhodospirillum rubrum according to any one of claims 1 to 9, for use in lowering LDL-cholesterol in the plasma of an individual.

12. The petroleum ether extract of rhodospirillum rubrum according to any one of claims 1 to 9, for use in a method of reducing LDL-cholesterol in the plasma of an individual.

13. Petroleum ether extract of rhodospirillum rubrum according to any of claims 1 to 9 for use according to claim 11 or 12, wherein the subject suffers from or has an increased risk of suffering from any one or more of the following diseases: cardiovascular disease, atherosclerosis, dyslipidemia, arteriosclerosis, hypercholesterolemia, familial hypercholesterolemia, hyperlipidemia, LDL plasma levels of at least 70mg/dL, total cholesterol levels of at least 200mg/dL, levels of lp (a) of at least 14mg/dL, inflammation, inflammatory disease, ischemia, infection.

14. The petroleum ether extract of rhodospirillum rubrum according to any one of claims 1 to 9, for the use according to any one of claims 11 to 13, wherein the extract is administered orally.

15. A food supplement having LDL-cholesterol lowering properties comprising a petroleum ether extract of rhodospirillum rubrum according to any one of claims 1 to 9.

16. A food product comprising the food supplement of claim 15.

17. A process for producing a petroleum ether extract of rhodospirillum rubrum, comprising the steps of:

a) providing rhodospirillum rubrum cells and providing a biphasic mixture of about 1:1 volume ratio of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol comprising about 0.03 wt% sodium chloride;

b) mixing the cells of step a) with the mixture of step a) at 15 to 25 ℃ for about 1 to 3 hours, preferably about 2 hours; and

c) separating the petroleum ether extract of rhodospirillum rubrum from the methanol comprising about 0.03 wt% sodium chloride, thereby providing the petroleum ether extract of rhodospirillum rubrum.

Background

Cardiovascular disease is caused by a number of synergistic factors, the most important of which is excessive blood cholesterol levels. Cholesterol is an essential building block for animal and human cells, since it is a component of cell membranes. Human cells can synthesize their own cholesterol, but cholesterol can also be absorbed from food. Both methods play an important role in cholesterol metabolism.

In addition to the basic biological role as a building block for cell membranes, cholesterol has negative effects on human health and is responsible for cardiovascular and cerebrovascular diseases (e.g. myocardial infarction, stroke and peripheral vascular disease), and more specifically is associated with the development of atherosclerotic lesions in the vessel wall. Elevated plasma cholesterol levels are the most important predictive risk factor for the development of cardiovascular and cerebrovascular disease and atherosclerosis.

In plasma, cholesterol is transported in so-called lipoproteins, which can be subdivided into a number of different classes, depending on their diameter and specific density. Very Low Density Lipoproteins (VLDL), Intermediate Density Lipoproteins (IDL), Low Density Lipoproteins (LDL) and High Density Lipoproteins (HDL) constitute the most important classes of lipoproteins.

Experimental and clinical studies have shown that the amount of cholesterol transported in VLDL, IDL and LDL lipoproteins (so-called atherogenic cholesterol) is a risk factor for the development of cardiovascular disease. In contrast, cholesterol transported in HDL particles prevents the development of cardiovascular and cerebrovascular diseases (anti-atherosclerotic cholesterol).

Randomized, placebo-controlled, prospective clinical studies have demonstrated that lowering plasma cholesterol has a beneficial effect on the morbidity and mortality of cardiovascular and cerebrovascular disease. However, it is a prerequisite that the reduction in cholesterol should be due primarily or substantially to a reduction in the atherogenic cholesterol present in VLDL, IDL and LDL, while the level of anti-atherosclerotic cholesterol preferably remains substantially unchanged.

Thus, for the treatment and prevention of cardiovascular disease, it is necessary to reduce the atherogenic cholesterol and increase the anti-atherosclerotic cholesterol in absolute or relative proportions.

A number of methods are available for reducing plasma cholesterol. Most importantly:

-inhibition of cholesterol biosynthesis;

-increasing the removal of cholesterol (and/or its metabolites, in particular bile acids) from the tissue into the intestinal lumen;

-reducing the absorption of cholesterol and bile acids by the gastrointestinal tract.

Drugs used to inhibit cholesterol synthesis are typically inhibitors of hydroxymethyl-glutaryl-coenzyme a reductase (HMGCoA reductase), the rate-limiting enzyme in the cholesterol synthesis pathway. These so-called "statins" are molecules that inhibit the action of enzymes. Examples are simvastatin, pravastatin and atorvastatin. Statins are generally chemically synthesized derivatives of naturally occurring fungal metabolites.

To increase cholesterol removal, bile acid adsorption resins (e.g., cholestyramine) may be used. As bile acids are adsorbed by the resin, their secretion in the faeces is increased and their resorption from the intestine into the blood is reduced, resulting in a relative loss of bile acids in the body. Thus, the liver increases the conversion of cholesterol to bile acids, resulting in a net increase in cholesterol secretion (metabolites) in the body. Since bile acids are essential for the uptake of cholesterol from the lumen into the intestinal tissue (by dissolving cholesterol), a reduction in the content of bile acids in the intestinal lumen also leads to a reduction in the cholesterol uptake.

Drugs that inhibit the active transport of cholesterol from the intestinal lumen to the blood by inhibiting the cellular transport system of cholesterol (and related sterols) in the intestinal epithelial cells are under development.

Cholesterol homeostasis

Maintenance of cholesterol homeostasis is vital to health and is achieved by a regulatory network consisting of genes involved in cholesterol synthesis, absorption, metabolism and elimination. An imbalance in cholesterol levels due to environmental and genetic factors leads to hypercholesterolemia, a major risk factor for atherosclerosis (i.e., hardening of arterial fouling) and related coronary and cerebrovascular diseases. Hypercholesterolemia and its associated cardiovascular disease are one of the largest global economic, social, and medical challenges we are now facing.

Despite the widespread use of therapeutic drugs for the control of blood cholesterol (such as statins that inhibit cholesterol synthesis), the fact remains that it is estimated that more than 50% of the us population has cholesterol levels at critical levels (i.e., >2 g/L). In addition, side effects associated with therapeutic drugs that control cholesterol levels have been reported, such as myopathy, liver damage, and potential drug-drug interactions. Therefore, the development of additional therapeutic agents for controlling cholesterol levels is essential, especially those drugs with better safety profile.

Chinese patent document CN1274584 discloses a rhodospirillum preparation for a diet food for reducing blood fat, and a method for producing the same.

Chinese patent document CN1172653 discloses a liquid rhodospirillum preparation for use in the treatment of the human digestive tract and a method for obtaining said preparation.

Japanese patent document JP08205819 discloses a culture medium for rhodospirillum and dried cells for rhodospirillum for use in beverages or foods for the treatment of diabetes, cardiac insufficiency.

However, none of these documents provides the following information: the specific fraction of cells and/or specific components of cells obtained after subjecting the cells to a detailed extraction protocol will be responsible for the claimed pharmaceutical activity.

Patent EP1569667 discloses the formulation of the membrane fraction of rhodospirillum, its use as a medicament, for use in food supplements, for use in food products, wherein the use is for lowering plasma cholesterol. Furthermore, EP1569667 discloses a method for providing membrane components of such rhodospirillum, comprising the steps of: culturing rhodospirillum cells into a multicellular culture; sonicating the cells; separating the membrane material from the cytoplasmic material by centrifugation; the precipitate recovered in the previous step was further washed and re-centrifuged.

However, there is still a need for cholesterol-lowering formulations, in particular for use in the food industry, and preferably aimed at human nutrition.

Summary of The Invention

Aspects of the present invention relate to a petroleum ether extract of rhodospirillum rubrum obtained by extracting cells of rhodospirillum rubrum with a mixture of petroleum ether having a boiling point of 60 to 80 ℃ and methanol containing sodium chloride.

Aspects of the present invention relate to a petroleum ether extract of rhodospirillum rubrum obtainable by extracting cells of rhodospirillum rubrum with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol comprising sodium chloride, wherein the extract is obtained by extraction at 8 ℃ to 37 ℃ for 10 minutes to 48 hours when the cells are mixed with the mixture, and wherein the cells of rhodospirillum rubrum are extracted with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol comprising sodium chloride in a volume ratio of 10:1 to 1: 10.

Preferred is a petroleum ether extract of rhodospirillum rubrum of the invention, wherein the extract is obtained by extraction at a temperature of 15 ℃ to 25 ℃ for 1 hour to 3 hours while mixing the cells with the mixture, and wherein the rhodospirillum rubrum cells are extracted with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol containing sodium chloride in a 1:1 volume ratio.

An aspect of the present invention relates to a petroleum ether extract of rhodospirillum rubrum according to the invention for use as a medicament.

An aspect of the present invention relates to a petroleum ether extract of rhodospirillum rubrum according to the invention for use in reducing LDL-cholesterol in the plasma of an individual.

Aspects of the invention relate to the petroleum ether extract of rhodospirillum rubrum of the invention for use in a method of reducing LDL-cholesterol in the plasma of an individual.

Aspects of the invention relate to petroleum ether extracts of rhodospirillum rubrum according to the invention for use in the treatment of any of cardiovascular disease, atherosclerosis, dyslipidemia, arteriosclerosis, hypercholesterolemia, familial hypercholesterolemia, hyperlipidemia, LDL plasma levels of at least 70mg/dL, total cholesterol levels of at least 200mg/dL, lp (a) levels of at least 14mg/dL, inflammation, inflammatory disease, ischemia, infection.

Aspects of the present invention relate to food supplements with LDL-cholesterol lowering properties comprising a petroleum ether extract of rhodospirillum rubrum of the present invention.

Aspects of the invention relate to a food product comprising the food supplement of the invention.

Aspects of the present invention relate to a method for producing a petroleum ether extract of rhodospirillum rubrum,

the method comprises the following steps:

a) providing rhodospirillum rubrum cells and providing a biphasic mixture of about 1:1 by volume of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol containing about 0.03% by weight of sodium chloride;

b) mixing the cells of step a) with the mixture of step a) at 18 ℃ to 24 ℃ for about 1 hour to 3 hours, preferably about 2 hours; and

c) separating the petroleum ether extract of rhodospirillum rubrum from methanol containing about 0.03 wt% sodium chloride, thereby providing the petroleum ether extract of rhodospirillum rubrum.

Brief Description of Drawings

Figure 1 shows the plasma cholesterol lowering effect on LDL-cholesterol in an in vivo animal model. Animals were fed control feed or feed enriched with extracts of rhodospirillum rubrum.

FIG. 2 Maldi TOF analysis of extracts of Rhodospirillum rubrum.

Detailed Description

The genus Rhodospirillum is a genus of the family Rhodospirillum, a family of purple non-sulfur bacteria of the order Rhodospirillum and α -Proteobacteria, among other characteristics, the family Rhodospirillum is characterized by being phototrophic, growing both aerobically and anaerobically, using light as an energy source for this purpose, bacteria contain chlorophyll b. within the genus Rhodospirillum, several species are distinguished, such as Rhodospirillum rubrum (Rhodospirillum rubrum), Rhodospirillum century (Rhodospirillum centenum), Rhodospirillum calometricum (Rhodospirillum phometicum), Rhodospirillum micum (Rhodospirillum rubrum), Rhodospirillum thiophilum (Rhodospirillum thiophilum), Rhodospirillum salina (Rhodospirillum solenium), Rhodospirillum halium and Rhodospirillum halodurans (Rhodospirillum sp. sp.and Rhodospirillum protothecium (Rhodospirillum).

Rhodospirillum rubrum can be found in natural water, mud neutralization sewage treatment plants, etc. Bacteria are used in sewage purification, for biomass production of animal food (e.g. as feed for poultry and fish) and as fertilizer. Due to the high vitamin and amino acid content, biomass of photooxygenation bacteria is considered to be an excellent raw material for animal feed.

The use of rhodospirillum rubrum as animal feed has been practiced for some time. It has been found that rhodospirillum rubrum cells can contribute significantly to the prevention of cardiovascular and cerebrovascular diseases by lowering the cholesterol level in plasma and/or serum (blood).

Cholesterol-lowering properties are defined herein as the ability of a composition, e.g., a cell extract, pharmaceutical composition, formulation, food supplement or food, to lower the cholesterol level or LDL-cholesterol level in the blood of an individual (e.g., an animal individual, such as a human individual) when administered to the body of the individual. Methods for measuring cholesterol levels in blood are known to the skilled person.

The first aspect of the present invention relates to a petroleum ether extract of rhodospirillum rubrum obtained by extracting cells of rhodospirillum rubrum with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol containing sodium chloride.

Other aspects of the present invention relate to a petroleum ether extract of rhodospirillum rubrum obtainable by extracting cells of rhodospirillum rubrum with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol comprising sodium chloride, wherein the extract is obtained by extraction at 8 ℃ to 37 ℃ for 10 minutes to 48 hours when the cells are mixed with the mixture, and wherein the cells of rhodospirillum rubrum are extracted with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol comprising sodium chloride in a volume ratio of 10:1 to 1: 10.

Throughout the specification, the term "petroleum ether having a boiling point of 60 ℃ to 80 ℃ has its conventional scientific meaning and herein refers to a petroleum component consisting of aliphatic hydrocarbons and boiling at 60 ℃ to 80 ℃.

A preparation of Rhodospirillum is defined herein as a cellular material of a quantity of Rhodospirillum which has been processed in some way. According to the present invention, the preparation of rhodospirillum is a petroleum ether extract with cholesterol lowering properties, such as a petroleum ether extract of rhodospirillum rubrum obtained according to the present invention by extracting cells of rhodospirillum rubrum with a mixture of petroleum ether having a boiling point of 60 to 80 ℃ and methanol containing sodium chloride.

The preparation according to the invention consists of a petroleum ether extract obtained from one species from the genus rhodospirillum, or the preparation according to the invention consists of a petroleum ether extract obtained from a mixture of different rhodospirillum species selected, for example, from rhodospirillum rubrum, rhodospirillum century, rhodospirillum calophyllum, rhodospirillum micranthum, rhodospirillum thiovorum, rhodospirillum salicornum, rhodospirillum halioti, rhodospirillum halitum and rhodospirillum gracilis.

The genus Phaeospirillum (Phaeospirillum) is another member of the family Rhodospirillaceae and includes Phaeospirillum fulvum, Phaeospirillum chandramohanii, Phaeospirillum oryzae (Phaeospirillum oryzae), Phaeospirillum tilakii and Phaeospirillum moliscianum. Thus, alternatively, the preparation according to the invention consists of a petroleum ether extract obtained from one species from the genus fuscoporia, or the preparation according to the invention consists of a petroleum ether extract obtained from a mixture of different fuscoporia fusca, selected from, for example, fuscoporia fulva, Phaeospirillum chandra mohanii, fuscoporia micans, Phaeospirillumtilakii and fuscoporia mowakami.

Of course, other alternative preparations according to the invention consist of petroleum ether extracts obtained from at least one species from the genus fuscoporia and at least one species from the genus rhodospirillum.

Preferably, the preparation of Rhodospirillum and/or Brospirillum nodosum comprises petroleum ether extracts of Rhodospirillum rubrum and/or Brospirillum morganii, more preferably of the species Rhodospirillum rubrum strain ATCC 11170 (strain DSM 467) or strain ATCC 25903 and/or Brospirillum morganii strain DSM 120(ATCC, American Type culture Collection), DSMZ, German culture Collection of microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen)).

The preparation of Rhodospirillum and/or Broussonetia according to the invention may contain 20-100% (w/w), preferably 40-100% (w/w), even more preferably 60-100% (w/w), and optimally 80-100% (w/w) of cell material from Rhodospirillum and/or Broussonetia, which is a petroleum ether extract of cells according to the invention. In addition, the formulation may contain other components depending on the method of preparation of the formulation selected. For example, the formulation may also contain water, or in the case of a lyophilized formulation, may also contain, for example, glycerol or sucrose or both.

In a preferred embodiment, a lyophilized formulation of rhodospirillum and/or fuscoporia fusca comprising a petroleum ether extract according to the invention is mixed with a filler material, such as microcrystalline cellulose (MCC) or mannitol, with a binder, such as hydroxypropyl cellulose (HPC), and/or a lubricant, such as stearic acid, and/or other excipients, and made into a dry powder or prepared for use in a different way.

The petroleum ether extract of rhodospirillum rubrum of the present invention is preferably a petroleum ether extract obtained by extraction at 15 ℃ to 25 ℃, preferably at about 18 ℃ to 24 ℃, more preferably at about room temperature for 1 hour to 3 hours, preferably about 2 hours, when the cells are mixed with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol containing sodium chloride.

Preferred is a petroleum ether extract of rhodospirillum rubrum according to the invention, wherein the extract is obtained by extraction at a temperature of 10 ℃ to 30 ℃, preferably 12 ℃ to 27 ℃, more preferably 15 ℃ to 25 ℃ for 20 minutes to 24 hours, preferably 30 minutes to 16 hours, more preferably 45 minutes to 8 hours, most preferably 1 hour to 3 hours, when mixing the cells with the mixture.

In an embodiment of the present invention, the petroleum ether extract of Rhodospirillum rubrum of the present invention obtained at room temperature for about 2 hours is extracted while mixing the cells with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol containing sodium chloride,

as used herein, the term "room temperature" has its conventional meaning and herein refers to a temperature of 20 ℃ to 22 ℃.

The petroleum ether extract of rhodospirillum rubrum of the present invention is preferably obtained by centrifuging the mixture at 18 ℃ to 24 ℃, preferably at about 20 ℃ to 22 ℃, more preferably at room temperature, for about 10 minutes to 60 minutes, preferably for about 20 minutes, after incubating the cells of rhodospirillum rubrum with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol containing sodium chloride.

Preferred is a petroleum ether extract of rhodospirillum rubrum according to the invention, wherein the cells of rhodospirillum rubrum are extracted with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol comprising sodium chloride in a volume ratio of 8:1 to 1:8, preferably 5:1 to 1:5, more preferably 2:1 to 1:2, most preferably 1: 1.

Therefore, the petroleum ether extract of rhodospirillum rubrum of the present invention is preferably obtained by extracting the cells of rhodospirillum rubrum with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol containing sodium chloride in about a 1:1 volume ratio, preferably 1:1 volume ratio.

Particularly preferred is a petroleum ether extract of rhodospirillum rubrum of the invention, wherein the extract is obtained by extraction at a temperature of 15 ℃ to 25 ℃ for 1 hour to 3 hours while mixing the cells with the mixture, and wherein the rhodospirillum rubrum cells are extracted with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol containing sodium chloride in a 1:1 volume ratio.

The petroleum ether extract of rhodospirillum rubrum of the present invention is preferably obtained by extracting the cells of said rhodospirillum rubrum with a mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol containing about 0.02% to 0.04% by weight of sodium chloride, preferably containing about 0.03% by weight of sodium chloride.

The petroleum ether extract of rhodospirillum rubrum of the invention preferably comprises one or more carotenoids and/or one or more quinones. Preferably, the petroleum ether extract of rhodospirillum rubrum of the invention comprises one or more carotenoids and one or more quinones.

The petroleum ether extract of rhodospirillum rubrum of the invention preferably comprises one or more carotenoids selected from the group consisting of the carotenoids rhodol, 1-hydroxy-spiroxanthin, 3, 4-didehydropurpurin b, chloroxanthin, bacteriopheophytin a, rhodoxanthin b, spiroxanthin, 3, 4-dihydrospiroxanthin and the phytyl derivatives of bacteriopheophytin a and/or one or more quinones selected from the group consisting of the following quinones: ubiquinol-10, ubiquinone-9, ubiquinone-10, and crimson quinone-10.

The petroleum ether extract of rhodospirillum rubrum of the present invention preferably comprises rhodoxanthin, 1-hydroxy-spiroxanthin, 3, 4-didehydropurpurin, chloroxanthin, bacteriopheophytin a, rhodoxanthin, spiroxanthin, 3, 4-dihydrospirillum, and phytylene derivatives of bacteriopheophytin a, ubiquinol-10, ubiquinone-9, ubiquinone-10, and profenoquinone-10.

Without wishing to be bound by theory, The biosynthesis of ubiquinone and erythroquinone from p-hydroxybenzoic acid and n-hydroxybenzaldehyde in Rhodospirillum rubrum is reported by Parson & Rudney in The Journal of biological chemistry (Vol.240, No. 4, 4 months 1965). The data presented by these authors indicate that growing rhodospirillum rubrum catabolizes ubiquinone to the derivative rhodoquinone.

According to the present invention, preferably, the petroleum ether extract of rhodospirillum rubrum of the invention comprises compounds having a molecular weight of at least 2100Da which in a NanoESI-mass spectrometry (NanoESI-MS) spectrum obtained with, for example, an ion trap NanoESI-MS high resolution triple time of flight mass spectrometer (Bruker) yields peaks having an m/z value of 752.7 and/or 769.3, said one or both peaks having an m/z value of 752.7 and/or 769.3 having an intensity in the NanoESI-MS spectrum which is at least two times the intensity of at least one, preferably two other peaks having an m/z value of 665.8 and 883.6 in the NanoESI-MS spectrum, preferably about five times said intensity.

According to the present invention, preferably, the petroleum ether extract of rhodospirillum rubrum of the invention comprises a compound having a molecular weight of at least 2100Da, which in the nanoESI-MS spectrum obtained with, for example, an ion trap nano-ESI-MS high resolution triple time of flight mass spectrometer (Bruker) yields peaks having m/z values of 752.7 and 769.3, said peaks having m/z values of 752.7 and 769.3 in the nanoESI-MS spectrum having an intensity at least twice, preferably about five times, the intensity of the other peaks having m/z values of 665.8 and 883.6 in the nanoESI-MS spectrum.

According to the present invention, preferably, nanoESI-MS analysis and measurement of the petroleum ether extract of Rhodospirillum rubrum of the present invention is carried out with said petroleum ether extract diluted in acetonitrile containing 1% v/v formic acid.

According to the present invention, preferably, the petroleum ether extract of rhodospirillum rubrum of the present invention comprises a compound producing one or more peaks, preferably three peaks, having m/z values of 500.3, 882.5 and 898.5 in matrix assisted laser desorption ionization time of flight (MALDI-ToF) Mass Spectrometry (MS) spectra, the intensity ratio of said one or more peaks having m/z values of 500.3, 882.5 and 898.5 in MALDI-ToF MS spectra being about 1:1:2, preferably about 1:1:3, more preferably about 1:1: 4.

According to the present invention, preferably, the petroleum ether extract of rhodospirillum rubrum of the present invention comprises a compound producing at least three peaks having m/z values of 500.3, 882.5 and 898.5, respectively, in the MALDI-ToF MS spectrum, the intensity ratio of said three peaks in the MALDI-ToF MS spectrum being about 1:1:2, preferably about 1:1:3, more preferably about 1:1:4, for m/z values 500.3, 882.5 and 898.5, respectively.

The aforementioned MALDI-ToF MS mass spectra are obtained, for example, with a Qtof Premier mass spectrometer (Waters) equipped with a Nd: YAG laser, operating at 355nm with an output frequency of 50Hz, with time-of-flight mass analysis performed in reflection mode with a resolution of about 10.000. The petroleum ether extract of the invention is then preferably analyzed using (DCTB) trans-2- [3- (4-tert-butylphenyl) -2-methylpropan-2-enylidene ] malononitrile matrix.

According to the invention, the previously described MALDI-ToF MS analysis and measurement of the petroleum ether extract of Rhodospirillum rubrum of the invention is preferably carried out with said petroleum ether extract dissolved in tetrahydrofuran.

According to the present invention, preferably, the petroleum ether extract of rhodospirillum rubrum of the present invention comprises a compound producing at least two peaks having m/z values of 651.6 and 647.6, respectively, in the nanoESI-MS spectrum obtained with, for example, a triple time of flight mass spectrometer (abciex), the two peaks having m/z values of 651.6 and 647.6, respectively, having an intensity ratio in the nanoESI-MS spectrum of about 1:1, preferably about 5:4, more preferably about 2: 1.

According to the present invention, preferably, the petroleum ether extract of rhodospirillum rubrum of the present invention comprises a compound producing at least two peaks having an m/z value of 881.5 and 927.5, respectively, in the nanoESI-MS spectrum obtainable with, for example, a triple time of flight mass spectrometer (abciex), the two peaks having an m/z value of 881.5 and 927.5, respectively, having an intensity ratio of about 1:1, preferably about 3:2, more preferably about 2:1, in the nanoESI-MS spectrum.

According to the present invention, preferably, the aforementioned nanoESI-MS analysis and measurement of the petroleum ether extract of Rhodospirillum rubrum of the present invention is carried out using an ABCIEX apparatus with said petroleum ether extract dissolved in a solution consisting of 99% v/v acetonitrile and 1% v/v formic acid.

Further aspects of the invention relate to a petroleum ether extract of rhodospirillum rubrum according to the invention for use as a medicament.

An aspect of the present invention relates to a petroleum ether extract of rhodospirillum rubrum according to the invention for use in reducing LDL-cholesterol in the plasma of an individual.

Aspects of the present invention relate to a petroleum ether extract of rhodospirillum rubrum according to the invention for use in a method of reducing LDL-cholesterol in the plasma of an individual. Similarly, an aspect of the invention relates to the use of a petroleum ether extract of rhodospirillum rubrum according to the invention for the preparation of a medicament for the treatment of high LDL-cholesterol in the plasma of an individual. Furthermore, an aspect of the invention relates to the use of a petroleum ether extract of rhodospirillum rubrum according to the invention for the preparation of a medicament for reducing LDL-cholesterol in the plasma of an individual.

Aspects of the invention relate to petroleum ether extracts of rhodospirillum rubrum according to the invention for use in the treatment of any of cardiovascular disease, atherosclerosis, dyslipidemia, arteriosclerosis, hypercholesterolemia, familial hypercholesterolemia, hyperlipidemia, LDL plasma levels of at least 70mg/dL, total cholesterol levels of at least 200mg/dL, lp (a) levels of at least 14mg/dL, inflammation, inflammatory disease, ischemia, infection.

In an embodiment of the invention, the use of a petroleum ether extract of rhodospirillum rubrum of the invention for the treatment of any one of cardiovascular disease, atherosclerosis, dyslipidemia, arteriosclerosis, hypercholesterolemia, familial hypercholesterolemia, hyperlipidemia, a LDL plasma level of at least 70mg/dL, a total cholesterol level of at least 200mg/dL, a lp (a) level of at least 14mg/dL, inflammation, inflammatory disease, ischemia, infection is for reducing LDL-cholesterol in the plasma of an individual suffering from, or at increased risk of suffering from, any of the above-mentioned diseases or health problems.

The preparation of rhodospirillum and/or the preparation of fusobacterium fuscois described above is a very suitable petroleum ether extract for use as a medicament or pharmaceutical preparation for lowering plasma cholesterol levels, preferably cholesterol levels in human plasma.

Preferably, the petroleum ether extract of rhodospirillum rubrum according to the invention is for use as a medicament according to the invention, wherein the use is for lowering LDL-cholesterol in the plasma of an individual.

In an embodiment of the invention, a petroleum ether extract of rhodospirillum rubrum is used for reducing LDL-cholesterol in the plasma of a subject, wherein said subject suffers from, or has an increased risk of suffering from, any one or more of the following diseases: cardiovascular disease, atherosclerosis, dyslipidemia, arteriosclerosis, hypercholesterolemia, familial hypercholesterolemia, hyperlipidemia, LDL plasma levels of at least 70mg/dL, total cholesterol levels of at least 200mg/dL, levels of lp (a) of at least 14mg/dL, inflammation, inflammatory disease, ischemia, infection.

When the petroleum ether extract of rhodospirillum rubrum of the invention is used in a method for reducing LDL-cholesterol in the plasma of an individual, the petroleum ether extract of rhodospirillum rubrum of the invention preferably reduces the level of LDL-cholesterol in the plasma, while the level of total cholesterol is substantially unchanged and/or while the level of HDL-cholesterol is substantially unchanged.

Surprisingly, the inventors of the present application found that the petroleum ether extract of rhodospirillum rubrum of the invention, for use as a medicament or pharmaceutical formulation for lowering plasma cholesterol levels, lowers LDL-cholesterol in plasma by at least 40%, such as about 46%, while at the same time the level of total cholesterol and the level of HDL-cholesterol are substantially unchanged.

According to the present invention, preferably, the petroleum ether extract of rhodospirillum rubrum of the invention for use as a medicament or pharmaceutical preparation for lowering plasma cholesterol levels lowers LDL-cholesterol in plasma by at least 20%, preferably about 40% to 80%, more preferably about 50%, while the levels of total cholesterol and HDL-cholesterol are substantially unchanged.

Preferably, the petroleum ether extract of rhodospirillum rubrum according to the invention for use according to the invention for reducing LDL-cholesterol in the plasma of an individual is administered orally.

Various embodiments of the formulation comprising a petroleum ether extract of rhodospirillum and/or fuscoporia fusca according to the invention are equally feasible. For example, the formulation of the invention may be provided as a fluid formulation of the invention containing solid components suspended, dispersed or emulsified in an aqueous solution, providing a composition according to the invention. Such inventive compositions may be used directly as a formulation comprising petroleum ether extracts of rhodospirillum and/or spirochaete fusca according to the invention, or may be processed into a food supplement in alternative embodiments.

Aspects of the present invention relate to food supplements with LDL-cholesterol lowering properties comprising a petroleum ether extract of rhodospirillum rubrum according to the invention.

In the present invention, a food supplement is defined as a formulation which can be consumed in addition to a normal diet and comprises components which are not present in the normal diet or are present in low or insufficient amounts, while sufficient or increased consumption of these components is required. Preferably, the food supplement according to the invention is configured such that it is suitable for human consumption. Thus, the food supplement as defined in the present invention should preferably have a texture, taste and smell that make the supplement suitable for human consumption.

In an embodiment of the invention, the food supplement with cholesterol lowering properties comprises a formulation of rhodospirillum and/or fuscoporia fusca comprising a petroleum ether extract of rhodospirillum and/or fuscoporia fusca according to the invention.

The food supplement according to the invention preferably contains from 0.1% to 99.9% (w/w) of a formulation of rhodospirillum comprising a petroleum ether extract of rhodospirillum. Preferably, the food supplement according to the invention contains from 10% to 90% (w/w), even more preferably from 30% to 75% (w/w) of a preparation of rhodospirillum and/or fusobacterium fuscogilum.

According to the present invention, in order to make a food supplement comprising a preparation of rhodospirillum and/or fuscoporia fusca containing a petroleum ether extract of rhodospirillum and/or fuscoporia fusca suitable for consumption, it is preferred to add a component that improves, for example, texture, taste or odor.

Thus, the food supplement according to the invention preferably comprises a (further) protein source, a carbohydrate source and a fat source, as well as vitamins, minerals, electrolytes, trace elements and other suitable components, making the food supplement itself suitable for use as a nutritional food.

Each protein and their mixtures suitable for use in nutritional formulations are preferably used in the food supplement according to the invention as a protein source. Such proteins encompass, for example, animal proteins (e.g. whey proteins, whey protein concentrates, whey powders, egg proteins, egg white proteins, casein or lactalbumin) and vegetable proteins (e.g. soy proteins, soy flour or proteins from soy milk). For selecting the protein source to be used, the biological value of the protein may constitute an important criterion. For example, caseinates, including calcium caseinate, but also whey, lactalbumin, ovalbumin and total egg protein are proteins of very high biological value because they contain a large proportion of essential amino acids.

For example, suitable carbohydrates to be used in the food supplement according to the invention are preferably simple short chain carbohydrates, such as mono-and disaccharides, but may also be polysaccharides, or a combination of both. According to the present invention, the carbohydrate is preferably selected for its suitable organoleptic properties. Preferably, according to the present invention, the complex carbohydrate is suitable for use as a dietary fibre.

In some embodiments, the food supplement according to the invention preferably contains a combination of simple carbohydrates and complex carbohydrates. In some embodiments, the food supplement according to the present invention preferably contains a fat selected from all edible oils and edible fats.

Vitamins and minerals are preferably added to the formulation according to the invention according to the provisions of the health authorities, and preferably cover all microorganisms and minerals recognized by the above authorities, such as vitamins A, B1, B2, B12, C, D, B and K, as well as folic acid, nicotinic acid, pantothenic acid and biotin. Such as iron, zinc, iodine, calcium, magnesium, chromium and selenium as minerals are preferably added to the formulation according to the invention.

Electrolytes (such as sodium, potassium and chloride ions) and trace elements and other additives also preferably form part of the food supplement according to the invention. Such components (if present) are preferably used at the recommended concentrations. In addition, the food supplement according to the invention preferably contains components improving its texture, colour and flavour, aroma substances, spices, fillers, emulsifiers, stabilizing compounds, preservatives, antioxidants, fibres and other supplements such as amino acids, choline, lecithin and fatty acids etc. The choice of such components depends on formulation, design and preference. The amount of such components added is known to the skilled person, and the selection of the amount to be added is preferably guided by considering the Recommended Daily Amount (RDA) for children and adults.

Emulsifiers are preferably added to stabilize the final product of the invention. Examples of acceptable emulsifiers according to the invention are lecithin (e.g. from soy or egg) and/or mono-and diglycerides. According to the invention, preference is given to using, for example, carob gum, guar gum or carrageenan as stabilizer.

Preservatives are preferably added to increase the shelf life of the product of the invention.

Preferably, preservatives are used in the formulations of the present invention, such as sodium sorbate, potassium benzoate, sodium benzoate, or calcium disodium EDTA.

According to the invention, natural or synthetic sweeteners (e.g. sugars, cyclamates, aspartame, acesulfame potassium and/or sorbitol) are preferably added to the food supplement in addition to the above mentioned carbohydrates.

The amount of the food supplement of the invention to be consumed varies in size and is not necessarily limited to the doses mentioned in the suggested dose. The term "food supplement" is not meant to be limited to a specified weight, or to a specified dosage of food supplement.

According to the invention, the composition of the food supplement according to the invention takes in principle any form suitable for human or animal consumption.

In a preferred embodiment of the invention, the food supplement is a dry powder suitable for suspension, dispersion or emulsification in aqueous solutions (e.g. coffee, tea, bouillon and juice). To this end, according to the invention, a dry powder is preferably provided in the dispenser.

In an alternative preferred embodiment of the invention, the food supplement is formulated as a tablet starting as a dry powder. To this end, preferably, the composition of the food supplement according to the invention is suitably provided with fillers (e.g. microcrystalline cellulose (MCC) and mannitol), binders (e.g. hydroxypropyl-cellulose (HPC)), lubricants (e.g. stearic acid) and other excipients.

The food supplement according to the present invention is preferably provided in an embodiment as a fluid in which the solid component has been suspended, dispersed or emulsified. Such compositions of the invention are preferably blended directly into the food product, or preferably, for example, extruded and pelletized or otherwise formed.

In an alternative embodiment of the invention, the food supplement is preferably formulated in a solid form, such as a bar, a biscuit or a roll.

The food supplement of the present invention is preferably formulated for oral consumption, preferably in combination with an acceptable carrier (e.g., capsule, tablet, water-soluble powder) or other form acceptable for administration. According to the present invention, it is alternatively preferred that the food supplement of the present invention is processed into a food product.

One aspect of the invention relates to a food product comprising the food supplement according to the invention.

Other aspects of the invention relate to a method of producing a formulation, food supplement or food product according to the invention.

Yet another aspect of the present invention relates to a method for producing a petroleum ether extract of rhodospirillum rubrum of the present invention, comprising the steps of:

(a) providing rhodospirillum rubrum cells and providing a biphasic mixture of petroleum ether having a boiling point of 60 ℃ to 80 ℃ and methanol containing about 0.03 wt% sodium chloride in a 1:1 volume ratio;

(b) mixing the cells of step a) with the mixture of step a) at 18 ℃ to 24 ℃, preferably at about 20 ℃ to 22 ℃, more preferably at room temperature, for about 1 hour to 3 hours, preferably about 2 hours; and

(c) separating the petroleum ether extract of rhodospirillum rubrum from methanol containing about 0.03 wt% sodium chloride, thereby providing the petroleum ether extract of rhodospirillum rubrum.

The method for producing the preparation according to the invention preferably comprises the steps necessary for culturing cells of one or more of rhodospirillum and/or fusobacterium fuscois to harvest said cells in said culture, and for processing the cells of said culture into the preparation.

Details of such methods are described in particular in the examples mentioned below. The skilled person will appreciate that various alternative methods may be used.

Anaerobic and phototrophic conditions are applied during the cultivation of cells of rhodospirillum and/or fuscoporia fusca. Various organic nutrients are preferably used as the carbon source. Very suitable media and growth conditions for cells of Rhodospirillum and/or Raspirillum fuscum are, for example, "Segers and Verstraete Medium" (Segers and Verstrae)te,1983) using lactic acid (about 2.7 g/L) as carbon source, pH about 6.8-6.9, and at a temperature of 25-37 deg.C, preferably under light from, for example, bar illumination (light intensity 300 μ M quantum. M. light)^-2.s^-1) Is adapted to the specific requirements of the microorganism concerned, under constant light intensity and anaerobic conditions. Other media suitable for the cultivation of Rhodospirillum and/or of Raspirillum fusca are, for example, "modified Rhodospirillaceae media" (DSMZ media #27, DMSZGmbH, Braunschweig, Germany) or Cens media (DSMZ media # 748). The cells are suitably cultured to a density of 0.01mg/mL to 50mg/mL, preferably 1mg/mL to 5mg/mL, based on the wet weight of the cells.

Cells were also grown anaerobically at 30 ℃ in 1 liter flasks containing a medium comprising 3.1ml/L of a 60% DL-lactate solution, 3g/L of bactopeptone, and 3g/L of yeast extract in tap water, the pH of the medium being 6.8, and 3W/L tungsten lamps at 50 μ M quantum. m.^-2.s^-1Is irradiated with the average intensity of the light radiation. After 3 days of growth, the optical density at 660nm amounted to 3.5(1.2g/kg dry weight).

Once the cells have reached the appropriate cell density, they are processed into the preparation according to the invention using separation from the growth medium, or harvesting by, for example, centrifugation or filtration.

After further processing, the concentrated cell mass is used as a preparation according to the invention.

Further steps of processing the cellular material of rhodospirillum and/or fusobacterium fuscois to obtain a useful preparation comprising the petroleum ether extract of rhodospirillum and/or fusobacterium fuscois of the invention include, for example, a washing step, and also further processing of the cells by extraction and optionally lyophilization.

Minnikin, O' Donnell et al describe a complete procedure for the extraction of bacterial isoprenoid quinones and polar lipids (Journal of Microbiological Methods 2(1984) pp.233-241). These authors outline the procedure for sequential extraction of isoprenoid quinones and polar lipids from bacterial cells. The upper phase containing isoprenoid quinone was extracted with a biphasic mixture of petroleum ether (b.p.60-80 deg.C) and methanol brine. As examples of the procedure, isoprenoid quinones of Bacillus subtilis (Bacillus subtilis), Mycobacterium avium (Mycobacterium avium), Pseudomonas defectives (Pseudomonas diminuta) and Streptomyces griseus (Streptomyces griseus) were extracted and analyzed.

Aspects of the present invention relate to petroleum ether extracts of rhodospirillum rubrum obtainable by the method of the invention.

The food supplement according to the invention may suitably be used to reduce intestinal cholesterol absorption and thereby reduce the cholesterol level in the plasma.

In another embodiment of the invention, the food supplement of the invention is used in a food product with cholesterol lowering properties.

The method of preparing the cholesterol-lowering food product of the invention comprises producing a food product incorporating the food supplement according to the invention. Such a method preferably comprises the steps wherein the food product is first prepared in the normal way and then the preparation of rhodospirillum and/or fusobacterium fuscois is added to the prepared food product. Furthermore, preparations of rhodospirillum and/or fusobacterium fuscogilum may be added to the food during the production of the food.

The food product with cholesterol lowering properties according to the invention typically contains 0.1% to 20% (w/w), preferably 1% to 10% (w/w) of the food supplement according to the invention and described above.

The invention finally comprises a formulation of rhodospirillum and/or fusspirillum fuscum comprising a petroleum ether extract of rhodospirillum and/or fuscoporia fuscum for use in a medicament for lowering plasma cholesterol levels, preferably plasma levels of LDL-cholesterol, while leaving the level of total cholesterol in the plasma substantially unchanged and/or while leaving the level of HDL-cholesterol in the plasma substantially unchanged. Preferably, such formulations of the invention comprising petroleum ether extracts of rhodospirillum and/or fusobacterium fuscois comprise the species rhodospirillum rubrum and/or fusobacterium morganii.

The invention is further illustrated by the following examples, which should not be construed as limiting the invention in any way.