Anti-hypercholesterolemia composition and method for producing the same

Abstract

The present invention relates to an antihypercholesterolemic composition and a method for preparing the composition. Accordingly, the present invention discloses a composition for reducing cholesterol formation and a method for preparing the composition. The antihypercholesterolemic composition contains an extract of Malabar Malusia leaf in the range of 30-70% by weight, an extract of red yeast rice in the range of 5-50% by weight, a squalene extract in the range of 1-30% by weight, a cordyceps sinensis extract in the range of 10-70% by weight, and at least one nutritionally or pharmaceutically acceptable excipient in the range of 0.01-50% by weight.

Description

Technical Field

[0001] The present invention relates to an anti-hypercholesterolemia composition and a method for producing the same. Background Art

[0002] Cholesterol is a waxy, fat-like substance found in all cells of the human body. Cholesterol is essential for the production of hormones, vitamin D and substances required for digestion. The cholesterol necessary for these functions is produced by the body or obtained from various food sources, particularly animal sources such as egg yolk, meat, and cheese. Broadly, cholesterol is classified into two categories: bad cholesterol (low-density lipoprotein-LDL) and good cholesterol (high-density lipoprotein-HDL). Generally, LDL deposits in blood vessels cause reduction in the size of blood vessels, impairing the flow of oxygen-rich blood throughout the body. Furthermore, LDL can also trigger the formation of blood clots, which often rupture and block blood flow, causing heart attacks and strokes. In contrast, HDL transports cholesterol from other parts of the body to the liver for removal from the body.

[0003] Although cholesterol is essential for the production of hormones, vitamin D and substances required for digestion, high concentrations of cholesterol combine with other substances in the blood to form plaques. Over a period of time, these plaques adhere to the walls of arteries and cause coronary artery disease. Therefore, lowering cholesterol levels is of utmost importance. Hypercholesterolemia is an elevated blood cholesterol level primarily caused by increased cholesterol concentrations in cells and plasma. It causes numerous cardiovascular diseases such as atherosclerosis, coronary heart disease, myocardial infarction and stroke.

[0004] There are many risk factors that can indicate a tendency towards high cholesterol levels, such as unhealthy eating habits and lifestyle, lack of exercise, genetics, age, weight, and health conditions such as diabetes, smoking, sex, race, and ethnicity. Cholesterol is primarily managed either through medication or through dietary changes. Of the medications, statins are generally the first-line treatment for hypercholesterolemia. However, despite being a first-line treatment, statins have a relatively low safety record and a high incidence of side effects. This leads to a decrease in long-term adherence to statins. Other cholesterol-lowering drugs, on the other hand, have a variety of side effects, including muscle pain and damage, liver damage, diabetes, confusion, and memory loss.

[0005] Therefore, there is a need for a composition that does not have the above-mentioned side effects and also has an anti-hypercholesterolemia effect. [Overview of the Initiative]

[0006] Accordingly, one aspect of the present invention provides a composition that reduces cholesterol formation. The anti-hypercholesterolemia composition contains an extract of Malabar spinach leaf in the range of 30-70% by weight, an extract of red yeast rice in the range of 5-50% by weight, an extract of squalene in the range of 1-30% by weight, an extract of Cordyceps in the range of 10-70% by weight, and at least one nutritionally or pharmaceutically acceptable excipient in the range of 0.01-50% by weight.

[0007] Another aspect of the present invention also discloses a method for producing an anti-hypercholesterolemia composition.

[0008] Examples of embodiments of the present invention may be shown in the accompanying drawings. These drawings are illustrative and not limiting. While the present invention outlines the circumstances of these embodiments, it should be understood that the scope of the present invention is not intended to be limited to these specific embodiments. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 shows the histopathological features of the heart: 1 shows the nucleus, 2 shows adipocytes, 3 shows the intercalation plate, 4 shows the myocardium, and A. normal control, B. high-fat diet (HFD) 1 ml / d + normal diet, C. high-fat diet (HFD) + normal diet + composition of the present invention (OD), D. high-fat diet (HFD) + normal diet + composition of the present invention (BD), E. high-fat diet (HFD) + normal diet + statin, and F. composition of the present invention (OD) given with a normal diet according to an embodiment of the present invention. [Figure 2] Figure 2 shows the histopathological features of the spleen: 1 shows the trabeculae, 2 shows the white pulp, 3 shows the red pulp, and A. Normal control. B. High-fat diet (HFD) 1 ml / d + normal diet. C. High-fat diet (HFD) + normal diet + composition of the present invention (OD), D. High-fat diet (HFD) + normal diet + composition of the present invention (BD), E. High-fat diet (HFD) + normal diet + statin, and F. Composition of the present invention (OD) given with a normal diet according to an embodiment of the present invention. [Figure 3] Figure 3 shows the histopathological features of the lung: 1 shows the bronchioles, 2 shows the alveoli, 3 shows the veins, and A. normal control, B. high-fat diet (HFD) 1 ml / d + normal diet, C. high-fat diet (HFD) + normal diet + composition of the present invention (OD), D. high-fat diet (HFD) + normal diet + composition of the present invention (BD), E. high-fat diet (HFD) + normal diet + statin, and F. composition of the present invention (OD) given with a normal diet according to an embodiment of the present invention. [Figure 4] Figure 4 shows the histopathological features of the kidney: 1 shows the plaque densa, 2 shows the proximal tubule, 3 shows the capillary, and 4 shows mesaglial cells. A. Normal control, B. High-fat diet (HFD) 1 ml / d + normal diet, C. High-fat diet (HFD) + normal diet + composition of the present invention (OD), D. High-fat diet (HFD) + normal diet + composition of the present invention (BD), E. High-fat diet (HFD) + normal diet + statin, and F. Composition of the present invention (OD) given with a normal diet according to an embodiment of the present invention. [Figure 5]Figure 5 shows the histopathological features of the liver: 1 shows the artery, 2 shows the bile duct, 3 shows the portal duct, 4 shows the portal vein, and A. normal control, B. high-fat diet (HFD) 1 ml / d + normal diet, C. high-fat diet (HFD) + normal diet + composition of the present invention (OD), D. high-fat diet (HFD) + normal diet + composition of the present invention (BD), E. high-fat diet (HFD) + normal diet + statin, and F. normal diet and composition of the present invention (OD) given according to embodiments of the present invention. [Figure 6] Figure 6 shows the histopathological features of the aorta, where 1 shows the intima, 2 shows the media, 3 shows the adventitia, and 4 shows the plaque. A. A normal rat, and B. A HED-fed rat showing changes in the intima with plaque formation in a cholesterol-induced group according to an embodiment of the present invention. [Figure 7] Figure 7 shows the histopathological image of the heart, where 1 shows the intercalation plate, 2 shows the nucleus, 3 shows the adipocyte, and 4 shows the myocardium. A. Normal control, B. Discontinuation of high-fat diet (HFD), C. Continuation of high-fat diet (HFD), D. Statin + high-fat diet (HFD) (discontinuation), E. Statin + high-fat diet (HFD) (continuation), F. Composition of the present invention (BD) + high-fat diet (HFD) (discontinuation), and G. Composition of the present invention (BD) + high-fat diet (HFD) (continuation) are according to embodiments of the present invention. [Figure 8] Figure 8 shows the histopathological features of the spleen: 1 shows the trabeculae, 2 shows the white pulp, 3 shows the central artery, 4 shows the red pulp, and A. normal control, B. discontinuation of high-fat diet (HFD), C. continuation of high-fat diet (HFD), D. statin + high-fat diet (HFD) (discontinuation), E. statin + high-fat diet (HFD) (continuation), F. composition of the present invention (BD) + high-fat diet (HFD) (discontinuation), and G. composition of the present invention (BD) + high-fat diet (HFD) (continuation) are according to embodiments of the present invention. [Figure 9] Figure 9 shows the histopathological features of the lung: 1 shows the bronchioles, 2 shows the alveoli, 3 shows the veins, and A. Normal control. B. Discontinuation of high-fat diet (HFD), C. Continuation of high-fat diet (HFD), D. Statin + high-fat diet (HFD) (discontinuation), E. Statin + high-fat diet (HFD) (continuation), F. Composition of the present invention (BD) + high-fat diet (HFD) (discontinuation), G. Composition of the present invention (BD) + high-fat diet (HFD) (continuation) according to an embodiment of the present invention. [Figure 10] Figure 10 shows the histopathological features of the kidney: 1 shows the plaque densa, 2 shows the proximal tubule, 3 shows the capillary, and 4 shows mesaglial cells. A. Normal control, B. High-fat diet (HFD) discontinued, C. High-fat diet (HFD) continued, D. Statin + high-fat diet (HFD) (discontinued), E. Statin + high-fat diet (HFD) (continued), F. Composition of the present invention (BD) + high-fat diet (HFD) (discontinued), G. Composition of the present invention (BD) + high-fat diet (HFD) (continued) according to an embodiment of the present invention. [Figure 11] Figure 11 shows the histopathological features of the liver: 1 shows the artery, 2 shows the bile duct, 3 shows the portal vein, 4 shows the portal duct, and A. normal control, B. discontinuation of high-fat diet (HFD), C. continuation of high-fat diet (HFD), D. statin + high-fat diet (HFD) (discontinuation), E. statin + high-fat diet (HFD) (continuation), F. composition of the present invention (BD) + high-fat diet (HFD) (discontinuation), G. composition of the present invention (BD) + high-fat diet (HFD) (continuation) according to an embodiment of the present invention. [Figure 12] Figure 12 shows the histopathological features of the aorta: 1 shows plaque, 2 shows the intima, 3 shows the media, 4 shows plaque breakdown, and 5 shows plaque regeneration. A. Normal control, B. High-fat diet (HFD) discontinued, C. High-fat diet (HFD) continued, D. Statin + high-fat diet (HFD) (discontinued), E. Statin + high-fat diet (HFD) (continued), F. Composition of the present invention (BD) + high-fat diet (HFD) (discontinued), G. Composition of the present invention (BD) + high-fat diet (HFD) according to an embodiment of the present invention. [Modes for carrying out the invention]

[0010] This invention discloses compositions that reduce cholesterol formation and methods for producing the same. One aspect of the invention disclosed is an anti-hypercholesterolemia composition. The composition contains an active ingredient that reduces cholesterol and at least one nutritionally or pharmaceutically acceptable excipient. In one embodiment of the invention, the cholesterol-lowering active ingredient is selected from extracts, fractions, active compounds, and vegetative chemicals or mixtures thereof from the group consisting of Malabar spinach, red yeast rice, Cordyceps sinensis, and squalene. These ingredients are obtained by conventional extraction procedures. In exemplary embodiments of the invention, the cholesterol-lowering active ingredient is an extract or an enriched fraction or a pure compound or mixture thereof.

[0011] Malabar spinach extract is obtained from Malabar spinach, also known as Malabar spinach, vine spinach, and Ceylon spinach. It is native to the Indian subcontinent, Southeast Asia, and New Guinea. However, it has naturalized in China, tropical Africa, Brazil, Belize, Colombia, the West Indies, Fiji, and French Polynesia, and as a result, the extract or raw material can be sourced from any of these various natural sources. Generally, plant extracts are derived from selected plant material from various parts of the plant, including but not limited to rhizomes, roots, stems, seeds, bark, flowers, leaves, and fruits, and are extracted using conventional extraction techniques with conventional solvents. In one embodiment of the present invention, the Malabar spinach extract is derived from the leaves of Malabar spinach. In one embodiment of the present invention, the Malabar spinach extract contains polyphenols in an amount ranging from 0.5 to 2%, phenols in an amount ranging from 0.5 to 2%, flavonoids in an amount ranging from 0.2 to 3%, and ascorbic acid in an amount ranging from 0.2 to 1%.

[0012] The scope of the present invention is not limited to Malabar spinach plants and products derived therefrom, but also extends to plants that are closely related botanically, particularly plants belonging to the same family, preferably plants belonging to the same genus, and more preferably plants belonging to the same species having substantially similar phenotypic and genotypic characteristics.

[0013] Conventional solvents are selected from the group consisting of water, alcohol, organic solvents, and combinations thereof, without limitation, or methods are selected from the group suitable for obtaining a complete extract, such as cold extraction, immersion, injection, decoction, permeation, high-temperature continuous extraction (Soxhlet), aqueous alcohol extraction, fermentation, countercurrent extraction, ultrasonic extraction (sonication), cold-press extraction, and supercritical fluid extraction. The extract may be solid, semi-solid, liquid, or nanoemulsion.

[0014] Red yeast rice is also known as red rice koji, red fermented rice, red koji rice, anka, or angkak. Red yeast rice is produced by fermenting cooked rice grains with Monascus mold, preferably Monascus purpureus, Monascus ruber, or Monascus pilosus. This includes all related strains of specific species such as Monascus purpureus went, Monascus purpureus NTU568, Monascus purpureus BCRC 3615, Monascus purpureus BCRC 31534, Monascus purpureus BCRC 31526, and Monascus purpureus MTCC 1090, which change the rice grains to a reddish-purple color due to their pigment-depositing ability. In one embodiment of the present invention, the monacolin K content in the red yeast rice is 0.1 to 100 ppm, and the ankaflavin content in the red yeast rice is 0.2 to 5%.

[0015] The scope of the present invention is not limited to Monascus fungi and products derived therefrom, but extends to microbiologically closely related microorganisms, particularly microorganisms belonging to the same family, preferably microorganisms belonging to the same genus, more preferably microorganisms belonging to the same species and strain having substantially similar phenotypic and genotypic characteristics.

[0016] Cordyceps is known as Chinese caterpillar fungus, Dong Chong Xia Cao, Caterpillar mushroom, Cs-4, Champignon chenille, Ophiocordyceps, or Vegetable caterpillar. Cordyceps is produced by a fermentation process, and is known to grow on cooked rice grains with a Cordyceps culture, preferably Cordyceps sinensis and Cordyceps militaris grown as a mat-like structure on rice grain medium or in suspension via a fermentation process, producing mycelia and further finger-like fruiting bodies resembling mushrooms. Cordyceps sinensis and Cordyceps militaris are native to Tibet, Nepal and India. However, they are commonly widespread throughout the Northern Hemisphere.

[0017] The scope of the present invention is not limited to Cordyceps fungi and products derived / extracted therefrom, but extends to other microbiologically closely related microorganisms, particularly microorganisms belonging to the same family, preferably microorganisms belonging to the same genus, more preferably microorganisms belonging to the same species and strain having substantially similar phenotypic and genotypic characteristics.

[0018] Squalene is an intermediate molecule in cholesterol biosynthesis, and is obtained from plant sources such as olives, soybeans, amaranth, rice bran, grape seeds, almonds, coconuts, palm and wheat germ. The sources are not limited to plants, and also include animal sources such as sharks, marine sources and animal-derived sources. In one embodiment of the present invention, the squalene extract is commercially available from the market.

[0019] In one embodiment of the present invention, the amount of the Basella alba leaf extract ranges from 30 to 70% by mass, the amount of the red yeast rice extract ranges from 5 to 50% by mass, the amount of the squalene extract ranges from 1 to 30% by mass, the amount of the Cordyceps sinensis extract ranges from 10 to 70% by mass, and the amount of the at least one nutritionally or pharmaceutically acceptable excipient ranges from 0.01 to 50% by mass.

[0020] In another embodiment of the present invention, the amount of the Basella alba leaf extract ranges from 35 to 55% by mass, the amount of the red yeast rice extract ranges from 5 to 20% by mass, the amount of the squalene extract ranges from 1 to 5% by mass, the amount of the Cordyceps sinensis extract ranges from 15 to 35% by mass, and the amount of the at least one nutritionally or pharmaceutically acceptable excipient ranges from 5 to 20% by mass.

[0021] In one embodiment of the present invention, the at least one nutritionally or pharmaceutically acceptable excipient is selected from the group consisting of at least one diluent, at least one superdisintegrant, at least one binder, at least one lubricant, at least one glidant, at least one filler, at least one vitamin, at least one mineral, at least one phytochemical, at least one antioxidant, and combinations thereof.

[0022] In one embodiment of the present invention, the at least one binder is selected from the group consisting of gelatin, ethyl cellulose, starch, polyvinylpyrrolidone, sodium alginate, carboxymethyl cellulose, silicon monoxide, Neusilin US2, dextrin, talc, magnesium stearate, Aerosil and microcrystalline cellulose.

[0023] In another embodiment of the present invention, at least one lubricant is selected from the group consisting of stearic acid, calcium stearate, sodium benzoate, and polyethylene glycol.

[0024] In yet another embodiment of the present invention, at least one lubricant is selected from the group consisting of corn starch and silicon dioxide.

[0025] In yet another embodiment of the present invention, at least one super-disintegrant is selected from the group consisting of lactose, microcrystalline cellulose, and sorbitol.

[0026] In embodiments of the present invention, the composition is prepared in a dosage form selected from the group consisting of semi-solid lumps, oils and water-soluble dispersions, nanoemulsions, capsules, tablets, syrups, blends, and suspensions. In other embodiments, the dosage form is further selected from the group consisting of immediate-release, sustained-release, and delayed-release. In exemplary embodiments of the present invention, the composition is encapsulated, and the dosage form is a capsule. Depending on the dosage, the composition may further contain excipients necessary for the production of a more preferred dosage form and for degradation after inoculation, which may be selected by those skilled in the art.

[0027] In yet another embodiment of the present invention, the capsule shell is prepared using a modified starch selected from the group consisting of corn starch, gelatin, HPMC, and carrageenan, without limitation.

[0028] Advantageously, the compositions of the present invention effectively reduce cholesterol formation and prevent LDL oxidation by reducing HMG-CoA reductase. The compositions of the present invention also remove existing plaque and reduce the incidence of plaque formation and atherosclerosis. Separately or in addition, the compositions may be used for the management, treatment, or prevention of hypercholesterolemia.

[0029] As an embodiment of the present invention, an anti-hypercholesterolemia composition for the treatment or prevention of hypercholesterolemia and atherosclerosis is disclosed. The composition contains 30 to 70% by mass of Malabar spinach leaf extract, 5 to 50% by mass of red yeast rice extract, 1 to 30% by mass of squalene extract, 10 to 70% by mass of Cordyceps sinensis extract, and 0.01 to 50% by mass of at least one nutritionally or pharmaceutically acceptable excipient.

[0030] In another aspect of the present invention, a method for producing the composition is disclosed. The method first includes obtaining an extract of Malabar spinach leaves, an extract of Cordyceps sinensis, an extract of red yeast rice, and an extract of squalene. Thereafter, predetermined amounts of these extracts are mixed with predetermined amounts of at least one nutritionally or pharmaceutically acceptable excipient to obtain the anti-hypercholesterolemia composition of the present invention.

[0031] In an embodiment of the present invention, an extract of Malabar spinach leaves is prepared by first obtaining a predetermined amount of Malabar spinach leaves and then drying them to remove moisture. The dried leaves are then ground to obtain ground plant material with a desired particle size that is most suitable for extraction. The ground plant material is then extracted using various solvents through various extraction processes, followed by the removal and recovery of the solvent by a rotary evaporator. The solvent used for extraction is selected from the group of ethanol, hexane, acetone, and redistilled water. The next step is nitrogen flushing to remove any residual solvent to obtain a crude extract. The crude extract is then dewaxed by dissolving the crude extract in a solvent and then deep freezing. Generally, ethanol, methanol, and hexane are used as solvents. Unwanted waxy substances are removed by cold filtering, followed by the addition of predetermined amounts of aerosil and dextrin to the crude extract, and then the solvent is removed by a rotary evaporator to obtain the Malabar spinach leaf extract in the form of a dry powder. The powder extract having a total flavonoid content exceeding 0.04% is stored in a dry, moisture-free state.

[0032] In another embodiment of the present invention, the red yeast rice extract is first prepared by grinding red yeast rice to obtain a powder. The powdered red yeast rice is then extracted using a suitable solvent in a grinder or homogenizer. The solvent is selected from the group consisting of water, ethanol, acetone, methanol, diethyl ether, and hexane. The obtained solvent extract is filtered. Generally, the solvent extraction and filtration steps are repeated 2 to 5 times to obtain the maximum extract. The obtained extract is left for 8 to 12 hours to obtain a red precipitate and a yellow suspended solid. The yellow suspended solid is then dried in a rotary evaporator to obtain a slurry. A drying excipient is then added to the slurry to obtain a powdered red yeast rice extract having a monacolin K content in the range of 0.1 ppm to 100 ppm.

[0033] In yet another embodiment of the present invention, the Monascus extract is prepared by first growing a predetermined amount of Monascus medium on rice grains, and then drying it to remove moisture. The dried rice grains fermented in the Monascus medium are then ground to obtain ground microbial material having a desired particle size suitable for maximum extraction. The powdered Monascus medium is extracted with acetone or ether or methanol or ethanol or dichloromethane or ethyl acetate or chloroform or hexane or water or a combination of solvents. This step is repeated 3 to 5 times to obtain the maximum extract. The extraction solvent is evaporated to dryness by a vacuum dryer or evaporator. The concentrated extract is recovered as a colored fraction by eluting it with a suitable solvent or a mixture thereof (acetone or ether or methanol or ethanol or dichloromethane or ethyl acetate or chloroform or hexane or water) through a silica gel or Sephadex or C18 column. To further purify the colored fractions, repeated column chromatography steps were performed using silica, Sephadex, or C18 column materials, and each fraction from the eluent was tested for monacin and ankaflavin content. Fractions rich in monacin and ankaflavin were recovered, and further purification was performed by preparative HPLC for high purity.

[0034] In one embodiment of the present invention, squalene extract is available directly from suppliers / markets as a value-added product. A common purification procedure for squalene from vegetable oil is silica-based column purification and elution with hexane.

[0035] In one embodiment of the present invention, Cordyceps extract is first prepared by growing a predetermined amount of Cordyceps culture medium on mycelium or fruiting bodies grown in a suitable culture medium. The fruiting bodies or mycelium are harvested, dried to remove moisture, and made into a powder of a desired particle size. A predetermined amount of the whole powder or extract is used for formulation purposes.

[0036] In one embodiment, the purity test of the components and the quantification method of the active ingredients are carried out using a spectrometer, gas chromatography, thin-layer chromatography, high-performance thin-layer chromatography, high-performance liquid chromatography, liquid chromatography, or mass spectrometry. [Examples]

[0037] (Example 1) Comparison of the potential of the composition of the present invention and statin (atorvastatin) to prevent atherosclerosis and hypercholesterolemia: Wistar albino rats weighing between 190 and 225 g were selected for this study. Temperature and humidity were maintained at optimal conditions (22+2°C, 40-70%), and the animals were exposed to a natural, actual day-night cycle. The experiment was conducted in accordance with the Animal Ethics Committee. (Preventive research) The 90-day preventive study used six rats per group. The study was conducted to determine the anti-hypercholesterolemia effects of statins (atorvastatin) and the composition of the present invention. The six groups were as follows: 1. Normal control 2.High fat diet (HFD)lml 1 / d+normal diet 3. High-fat diet (HFD) + regular diet + composition of the present invention (OD) 4. High-fat diet (HFD) + regular diet + composition of the present invention (BD) 5. High-fat diet (HFD) + regular diet + statins 6. Composition of the present invention (OD) given together with a regular meal The treatment study was conducted after inducing cholesterol and atherosclerosis with HFD for 90 days, and after 60 days of treatment, the efficacy of the composition of the present invention was investigated in comparison to statins. Body weight was recorded to the nearest gram (g) with the subject placed on a balance in a steady state and using an appropriate tare weight to 0.0000. The same weighing machine was used for all subjects. The machine was tested for several errors with a known set of weights.

[0038] [Table 1] [Table 2] [Table 3]

[0039] [Table 4] [Table 5] [Table 6]

[0040] The data summarized in Tables 1-6 show the mean and standard deviations of lipid profiles and hematological parameters, indicating that the composition of the present invention was able to maintain body weight even after HFD treatment, while statins were not. Furthermore, the reduction in total cholesterol was similar in both groups (the composition and the statin group) even after HFD diet treatment. However, significant suppression was observed in total cholesterol synthesis (75%), LDL synthesis (85%), and triglycerides (50-70%). Apart from hemoglobin, it increased by 15% with the composition of the present invention administered twice daily, and no significant changes were observed in blood parameters. (Biochemical analysis) 1.5-2 ml blood samples were collected from the retino-orbital chamber in vials and biochemically evaluated every 15 days. The tests performed included total cholesterol (TC) (mg / dl), triglycerides (TG) (mg / dl), high-density lipoprotein (HDL-C) (mg / dl), low-density lipoprotein (LDL-C) (mg / dl), very low-density lipoprotein (VLDL) (mg / dl), and WBC (10) 3 / ul), RBC(10 6 / ul), Hb (g / dl), PCV (%), red blood cells (%), platelet count 10 5 / mm3 This includes details of coagulation time (seconds), neutrophils, lymphocytes, monocytes, and eosinophils. Assessments were performed using the RX50 semi-autoanalyzer from Erba Mannhelm, ERBA diagnostics Mannhelm, MicroLabs Instruments, Germany, and the COUNCELL21 Ecoline kit from Tulip Diagnostics Pvt. Ltd. Lipid oxidation reactions (plasma and liver), HMG-Co reductase, creatine phosphokinase (CPK), lactate dehydrogenase (LDH), SGOT, and SGPT were estimated at the end of both studies using the kit.

[0041] (Analysis of HMG-CoA reductase activity) Tissue homogenates were prepared by homogenizing 1 g of tissue (liver) with 10 ml of arsenate solution. Equal volumes of fresh 10% tissue homogenate and dilute perchloric acid were then mixed, allowed to stand for 5 minutes, and centrifuged (2000 rpm, 10 minutes). Next, 1.0 ml of the filtrate was treated with 0.5 ml of freshly prepared hydroxyamine reagent (alkaline hydroxyamine reagent in the case of HMG-CoA) and mixed. After 5 minutes, 1.5 ml of ferric chloride reagent was added to the same tube and shaken well. Measurements were obtained after 10 minutes against a similarly treated saline / arsenate blank at 540 nm.

[0042] [Table 7]

[0043] The data shown in Table 7 represent enzyme estimates after euthanasia of the animals. It is clear that HFD treatment induced a 20% reduction in hepatic lipid oxidation reactions, which was significantly reduced by 23% in the group receiving the composition of the present invention twice daily and significantly reduced by 15% in the group receiving statin treatment. Creatine phosphokinase activity was high in both HFD groups, and muscle damage was observed in the statin-treated groups (15-20%). Inhibition of SGOT (24%) and SGPT (29%) activity was most pronounced in the group receiving the composition of the present invention twice daily compared to statins (15% and 20%). Both the composition of the present invention and statin treatment allowed for similar blood glucose levels to the normal diet group, even in the group given HFD later. Inhibition of HMG Co reductase activity by the composition of the present invention and statins was similar, i.e., 35%.

[0044] (statistical analysis) Statistical analyses within and between groups were performed using SPSS software, comparing initial and final values. The mean + SD (standard deviation) and p-values ​​for all clinical parameters were calculated using paired t-tests in a 2x2 contingency table. All groups were compared using Pearson's chi-squared test. The obtained data were also analyzed using analysis of variance (ANOVA) and multivariate analysis, followed by Dunnett's multi-comparative test and Tukey-Kramer's test to determine the level of significance of observed effects. All p-values ​​were two-tailed, and a difference of P < 0.05 was considered statistically significant; all significant data suggest a strong association with the clinical parameter. All groups were compared using Pearson's chi-squared test.

[0045] (Example 2) Histopathological analysis Figure 1 shows the histopathological features of the heart. Histopathological findings in preventive studies of the heart showed a significant amount of fat deposition in the myocardium of HFD-induced rats compared to the hearts of normal rats. On the other hand, in the case of statins, the myocardium showed relaxation as the fat content decreased. However, rats treated with the organism of the present invention also showed optimal recovery of myocardial morphology, even during HFD treatment. In conclusion, the composition of the present invention showed better results compared to all groups. Figure 2 shows the histopathological findings of the spleen. The histopathological findings in the spleen in the preventive studies showed no significant changes in any of the study groups. However, this demonstrates the safety of the composition of the present invention in the spleen. Figure 3 shows the histopathological findings of the lungs. Histopathological findings in the lungs of various groups in the preventive study showed no pathological changes in any of the study groups. However, this demonstrates the safety of the composition of the present invention in the lungs. Figure 4 shows the histopathological findings of the kidney. Histopathological findings in the kidneys of various groups in the preventive study showed no pathological changes in any of the study groups. However, this demonstrates the safety of the composition of the present invention in the kidney. Figure 5 shows the histopathological features of the liver. Histopathological findings in the liver in preventive studies showed significant adipocyte formation (grade 2) in HFD-induced rats. However, intervention with the composition of the present invention at OD and BD administration levels showed a reduction in the ranking of fatty liver from grade 2 to grade 1 (as evidenced by dilation of the hepatic portal vein triplica). In the statin group, dilation of the hepatic portal vein triplica was more pronounced compared to the composition of the present invention (BD administration). The safety of the composition of the present invention in the liver was also demonstrated. The composition of the present invention was also found to be useful in preventing the deposition of fat bodies.

[0046] The experiment showed typical results, with normal weight gain. In the high-fat diet (HFD) 1 ml / d and normal diet groups, body weight and lipid profile parameters showed significant increases compared to the normal control group (P<0.005). There were no major changes recorded between the high-fat diet (HFD), normal diet, and the composition of the present invention (OD), and the profiles were almost identical to those of the high-fat diet 1 ml / d and normal diet groups. However, the high-fat diet, normal diet, and composition of the present invention (BD) showed a dramatic reversal of biochemical parameters, similar to the high-fat diet (HFD), normal diet, and statin groups, and showed a significant association (P<0.005). Furthermore, the composition of the present invention (OD), given with a normal diet, also showed significant changes in biochemical profiling compared to the normal control group and was found to be almost identical to the statin group. In multivariate analysis, the data suggested that the composition of the present invention (BD) showed an improved effect compared to the statin group (P=0.070 and 0.058, respectively). Statistically, the aforementioned data showed a significant correlation in biochemical and clinical profiling from day 0 to day 90 (P<0.001). Thus, the composition of the present invention showed a significant cholesterol-lowering effect even in a group of Wistar rat models with hypercholesterolemia-induced cholesterol, i.e., under continuous high-fat diet treatment, showing results similar to those of statins. Furthermore, the composition of the present invention at an oral dose of 82.5 mg / kg body weight (OD&OB) showed no signs of toxicity or death throughout the entire study period and was found to be as safe as in animals in a house environment. Figure 7 shows the histopathological findings of the heart. Histopathological findings in the hearts of the group continuously fed the HFD diet in the preventive study showed morphological changes (fat deposition) in the myocardium compared to the hearts of normal rats. Intervention using the composition of the present invention showed a significant improvement in HFD-induced morphological changes compared to the statin group. In the HFD-C group, the myocardium was found to be stressed due to high fat deposition in the statin group, whereas a significant improvement was evident in the group given the composition of the present invention.

[0047] Figure 8 shows the histopathological features of the spleen. Histopathological findings in spleen prevention studies across various groups showed no pathological changes in any of the study groups, leading to the conclusion that the composition of the present invention is safe in the spleen. Figure 9 shows the histopathological findings of the lungs. Histopathological findings in lung prevention studies in various groups showed no pathological changes in any of the study groups, leading to the conclusion that the composition of the present invention is safe in the lungs. Figure 10 shows the histopathological features of the kidney. Histopathological findings in various groups of kidney prophylaxis studies showed no pathological changes in any of the study groups, leading to the conclusion that the composition of the present invention is safe in the kidney. Figure 11 shows the histopathological findings of the liver. Histopathological findings in the liver in the preventive study showed significant formation of adipocytes (grade 3) in HFD-induced rats (continued), but intervention with statins and the composition of the present invention showed greater recovery in the group given the composition of the present invention (as evidenced by dilation of the hepatic portal vein triduct) compared with the statin group. Furthermore, the composition of the present invention improved the histopathological findings of fatty liver from grade 3 to grade 1.

[0048] (Example 3) Lipid profile and hematological analysis

[0049] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14]

[0050] Tables 7 to 12 suggest that the reductions in total cholesterol, LDL, and TG were significantly higher in the group using the composition of the present invention compared to the statin group (13%, 8%, and 11%), namely 22%, 17%, and 26%, respectively. HDL levels also improved in the group using the composition of the present invention compared to the statin group, namely 44 mg / dl vs. 39 mg / dl.

[0051] [Table 15]

[0052] Table 11 clearly shows that the reduction in liver LPO with the composition of the present invention was 18%, compared to 11% with statins. The inhibition of HMG CO reductase activity by the composition of the present invention and statins was similar, at 35%. The reduction in SGOT with the composition of the present invention was higher at 22%, compared to 16% with statins. The reduction in serum glutamate pyruvate transaminase (SGPT) was significantly higher with the composition of the present invention, at 23%, compared to 5% with statins.

[0053] (Example 4) Histopathological analysis of the aorta Figures 6 and 12 show the histopathological findings of rat aortas. Histopathological findings in plaqued aortas after discontinuation of a high-fat diet indicate the successful development of a rat model of atherosclerosis, and more severe plaque formation was observed in the group that continued the high-fat diet. Statin intervention after discontinuation of the high-fat diet showed a moderate reduction in plaque, while statin intervention while continuing the high-fat diet showed only a mild reduction. The morphology of the group that ingested the composition of the present invention showed almost complete plaque reduction / degradation similar to that of a normal rat aorta. Examples 1-4 demonstrated the most important finding in the preventive studies summarized above regarding the mean and standard deviation of lipid profiles and hematological parameters: the composition of the present invention was able to maintain body weight even after HFD treatment, whereas statins were not. Furthermore, although the increase in TC was similar (10%) in both the composition of the present invention and the statin group even after HFD diet, it was clear that HFD treatment induced hepatic lipid peroxidation by 20%, which was significantly reduced (23%) in the group given the composition of the present invention (twice daily) and by 15% with statin treatment. Creatine phosphokinase activity was also detected at high levels in both the HFD and statin-treated groups (8%-10%) that showed muscle damage, whereas no muscle damage was reported in the group given the composition of the present invention.

[0054] Furthermore, inhibition of SGOT (24%) and SGPT (29%) activity was maximized in the group treated with the composition of the present invention (twice daily), whereas the other groups were unable to control SGPT and SGOT levels. However, both the composition of the present invention and statin treatment were able to maintain blood glucose levels similar to those of a normal diet, even after the group given HFD.

[0055] Furthermore, histopathological findings in preventive studies of the heart showed distinct fat deposition in the myocardium of HFD-induced rats compared to the hearts of normal rats. In contrast, with statins, the myocardium showed relaxation as the fat content decreased. Moreover, rats treated with the composition of the present invention showed improvement or better recovery of myocardial morphology even during treatment with HFD. Histopathological findings of the liver showed significant adipocyte formation (grade 2) in HFD-induced rats, but intervention with the composition of the present invention at statin and OD and OB dose levels showed a reduction in fatty liver from grade 2 to grade 1 (evidence of dilation of the hepatic portal vein triduct). No significant changes were observed in all other groups. Statistically, the data showed a significant correlation with biochemical and clinical profiling from day 0 to day 90 (P<0.001). These examples demonstrate that the composition of the present invention has a significant cholesterol-lowering effect on a hypercholesterolemia-induced Wistar rat model, even in a group fed a continuously high-fat diet, and showed improved results compared to statins (atorvastatin).

[0056] Examples 1-4 demonstrate that in therapeutic studies, the reductions in total cholesterol, LDL, and TG were significantly higher in the groups treated with the compositions of the present invention (22%, 17%, and 26%) compared to the statin groups (13%, 8%, and 11%, respectively). Furthermore, anthropometric and biochemical data of normal control rats typically show a 10% weight gain over the entire 60-day laboratory study. In the group that continued on a high-fat diet (HFD), weight and lipid profile parameters showed a significant increase in the visceral pouch and were significantly lethargic compared to normal healthy controls.

[0057] Furthermore, the inhibition of HMG Co reductase activity by the composition of the present invention and statins was found to be similar, i.e., 35%. The reduction in SGOT was higher at 22% compared to 16% in the statin group. The reduction in SGPT was significantly higher at 23% in the group treated with the composition of the present invention compared to the statin group, i.e., 5%. These examples demonstrate that the composition of the present invention has a significant effect on atherosclerosis and has an anti-hypercholesterolemia effect in a hypercholesterolemia-induced Wistar rat model.

[0058] The examples showed that the composition of the present invention, administered orally at a dose of 135 mg / kg body weight (BD), showed no signs of toxicity or death and was found to be safe for animals in a house environment. Blood parameters / markers correlated with histopathological analysis as follows: Histopathological findings of the liver showed significant adipocyte (grade 3) formation in HFD-induced rats (continued), but intervention with statins and the composition of the present invention showed greater recovery in the group given the composition of the present invention (as evidenced by dilation of the hepatic portal vein triduct) compared to the statin group. Furthermore, the group given the composition of the present invention improved the histopathology of fatty liver from grade 3 to grade 1. Histopathological findings in the aorta after discontinuation of the high-fat diet showed that plaque was present, indicating the successful development of an atherosclerotic rat model, and more severe plaque formation was observed in the HFD continuation group. HFD discontinuation and statin intervention showed a moderate reduction in plaque, while HFD continuation and statin showed only a mild reduction. The morphology of the group given the composition of the present invention showed almost complete plaque reduction / degradation, similar to that of a normal rat aorta. Histopathological findings also showed plaque formation after high-fat diet treatment. Similar plaques were observed to be completely removed / dissolved after treatment with the composition of the present invention.

[0059] Histopathological studies showed no pathological changes in the intima of the normal group. The control group (high-fat diet) showed severe intima damage. The group treated with the composition of the present invention showed less intima damage compared to the HFD group, while the group treated with atorvastatin showed mild intima damage. Atherosclerosis was induced in the HFD group, and the composition of the present invention restored it to a normal state as shown in histological studies. Furthermore, vitamin D deficiency was also associated with treatment with atorvastatin (35 IU), but the composition of the present invention (44 IU) maintained healthy vitamin D levels in plasma compared to the control (42 IU). Therefore, the associated side effects are associated with regular statin treatment for 60 days.

[0060] Furthermore, the reduction in liver LPO with the composition of the present invention was 18%, compared to 11% with statins. Inhibition of HMG Co reductase activity by the composition of the present invention and statins was similar, at 35%. The reduction in SGOT was higher at 22% compared to 16% with statins. The reduction in SGPT was significantly higher in the group given the composition of the present invention (23%) compared to the statin group (5%).

[0061] Furthermore, the examples also highlight liver damage / fatty liver associated with routine statin treatment, which correlates with histopathological analysis. CPK levels were found to increase by 10% in the statin group, reflecting muscle damage. The composition of the present invention does not raise CPK levels. Muscle damage is also associated with pain such as muscle fatigue caused by statin treatment. Sodium and potassium levels were elevated in the statin group, but the composition of the present invention was also found to maintain Na (sodium) and K (potassium) levels. Prolonged elevations of electrolytes such as Na are associated with blood pressure disorders affecting the heart. Thus, the composition of the present invention is more effective than statins in terms of its potential to anti-atherosclerosis and anti-hypercholesterolemia, and concentrations of 82.5 mg / kg body weight (OD&BD) and 135 mg / kg body weight (BD) in oral doses of the composition of the present invention were shown to be safe in both prophylactic and therapeutic studies, showing no signs of toxicity or death. Furthermore, histopathological and blood marker studies have shown that statin side effects such as vitamin D deficiency, liver damage, Na and K levels, and muscle fatigue are not associated with long-term use of the compositions of the present invention.

[0062] Although the present invention has been described in terms of specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the scope of the invention as defined in the claims. Another aspect of the present invention may be as follows: 〔1〕 -30-70% by mass of Malabar spinach leaf extract, -5 to 50% by mass of red yeast rice extract, -Squalene extract in the range of -1 to 30% by mass, Cordyceps extract in the range of -10 to 70% by mass, and An anti-hypercholesterolemia composition containing at least one nutritionally or pharmaceutically acceptable excipient in the range of -0.01 to 50% by mass. [2] The composition according to [1], wherein the at least one nutritionally or pharmaceutically acceptable excipient is selected from the group consisting of diluents, super-disintegrants, binders, lubricants, lubricants, fillers, vitamins, minerals, vegetative chemicals, and antioxidants. [3] The composition according to [2], wherein the at least one binder is selected from the group consisting of gelatin, ethylcellulose, starch, polyvinylpyrrolidone, sodium alginate, carboxymethylcellulose, silicon monoxide, Neusilin US2, dextrin, talc, magnesium stearate, Aerosil, and microcrystalline cellulose. [4] The composition according to [2], wherein the at least one lubricant is selected from the group consisting of stearic acid, calcium stearate, sodium benzoate, and polyethylene glycol. [5] The composition according to [2], wherein the at least one lubricant is selected from the group consisting of corn starch and silicon dioxide. [6] The composition according to [2], wherein the at least one super-disintegrant is selected from the group consisting of lactose, microcrystalline cellulose, and sorbitol. 〔7〕 - The vitamins are selected from the group of vitamins D, C, E, A, and K. -Minerals are selected from the group of zinc, calcium, magnesium, iron, and selenium. - The vegetation chemical is curcumin, and - The composition according to [2] above, wherein the antioxidant is selected from the group consisting of quercetin and resveratrol. [8] The Malabar spinach extract is - A quantity of polyphenols in the range of -0.5 to 2% - A phenol amount in the range of -0.5 to 2% - Flavonoids in amounts ranging from -0.2 to 3%, and The composition according to [1] above, containing ascorbic acid in an amount ranging from -0.2% to 1%. [9] The composition according to [1], wherein the monacolin K content in the red yeast rice extract is 0.1 to 100 ppm, and the ankaflavin content in the red yeast rice extract is 0.2 to 5%. 〔10〕 - A step of preparing an extract of Malabar spinach leaves, an extract of Cordyceps sinensis, an extract of red yeast rice, and an extract of squalene, The extract of the aforementioned Malabar spinach leaves is as follows: The process involves drying a predetermined amount of Malabar spinach leaves, and then grinding them to obtain ground plant material having a desired particle size. The process involves extracting the ground plant material using a solvent selected from ethanol, hexane, acetone, and redistilled water, followed by the removal and recovery of the solvent. • A step to obtain a crude extract by nitrogen flushing to remove residual solvent. The crude extract is dissolved in at least one of ethanol, methanol, and hexane, and then dewaxed by deep freezing. The preparation involves cold filtering to remove unwanted waxy substances, followed by adding predetermined amounts of aerosil and dextrin to the crude extract to obtain an extract of Malabar spinach leaves. The Cordyceps sinensis extract is as follows: - A step of growing a predetermined amount of Cordyceps in the form of mycelium or fruiting bodies, and The preparation involves the process of collecting the fruiting body or mycelium, drying it, and then powdering it to obtain Cordyceps sinensis extract. The extract of the aforementioned red yeast rice is as follows: • The process of grinding red yeast rice to obtain a powder. The process involves grinding or homogenizing the material using a solvent selected from the group consisting of water, ethanol, acetone, methanol, acetone, diethyl ether, and hexane, followed by filtration to obtain an extract. - A step of maintaining the extract for 8 to 12 hours to obtain a red precipitate and a yellow suspended solid. • A process of drying the yellow suspended material to obtain a slurry. The preparation is carried out by the step of adding a dry excipient to the slurry to obtain a powdered red yeast rice extract. And, A method for producing an anti-hypercholesterolemia composition, comprising the step of mixing an extract with a predetermined amount of at least one nutritionally or pharmaceutically acceptable excipient to obtain an anti-hypercholesterolemia composition. 〔11〕 - Extract of Malabar spinach leaves in the range of 30-70% by weight; -5 to 50% by weight of red yeast rice extract; - Squalene extract in the range of -1 to 30% by weight; Cordyceps extract in the range of -10 to 70% by weight; and An anti-hypercholesterolemia composition for the treatment or prevention of hypercholesterolemia and atherosclerosis, comprising at least one nutritionally or pharmaceutically acceptable excipient in the range of -0.01 to 50% by weight.

[12] The composition according to

[11] , wherein the at least one nutritionally or pharmaceutically acceptable excipient is selected from the group consisting of diluents, super-disintegrants, binders, lubricants, fillers, vitamins, minerals, vegetatives and antioxidants.

Claims

1. - Extract of Malabar spinach leaves in the range of 30 to 70% by mass, - Red yeast rice extract in the range of 5 to 50% by mass, - Squalene extract in the range of 1 to 30% by mass, Cordyceps powder in the range of -10 to 35% by mass, and - An anti-hypercholesterolemia composition containing at least one nutritionally or pharmaceutically acceptable excipient in an amount ranging from 0.01 to 50% by mass.

2. The composition according to claim 1, wherein the at least one nutritionally or pharmaceutically acceptable excipient is selected from the group consisting of diluents, super-disintegrants, binders, lubricants, fillers, vitamins, minerals, vegetative chemicals, and antioxidants.

3. The composition according to claim 2, wherein the at least one binder is selected from the group consisting of gelatin, ethylcellulose, starch, polyvinylpyrrolidone, sodium alginate, carboxymethylcellulose, silicon monoxide, Neusilin US2, dextrin, talc, magnesium stearate, Aerosil, and microcrystalline cellulose.

4. The composition according to claim 2, wherein the at least one lubricant is selected from the group consisting of stearic acid, calcium stearate, sodium benzoate, and polyethylene glycol.

5. The composition according to claim 2, wherein the at least one lubricant is selected from the group consisting of corn starch and silicon dioxide.

6. The composition according to claim 2, wherein the at least one super-disintegrant is selected from the group consisting of lactose, microcrystalline cellulose, and sorbitol.

7. - The vitamins are selected from the group of vitamins D, C, E, A, and K. -Minerals are selected from the group of zinc, calcium, magnesium, iron, and selenium. - The vegetation chemical is curcumin, and - The composition according to claim 2, wherein the antioxidant is selected from the group consisting of quercetin and resveratrol.

8. The aforementioned Malabar spinach extract - A quantity of polyphenols in the range of -0.5 to 2% - A quantity of phenols ranging from 0.5% to 2% - Flavonoids in amounts ranging from 0.2% to 3%, and The composition according to claim 1, comprising an amount of ascorbic acid in the range of -0.2 to 1%.

9. The composition according to claim 1, wherein the monacolin K content in the red yeast rice extract is 0.1 to 100 ppm, and the ankaflavin content in the red yeast rice extract is 0.2 to 5%.

10. A method for producing an anti-hypercholesterolemia composition, - A process for preparing an extract of Malabar spinach leaves, Cordyceps sinensis powder, red yeast rice extract and squalene extract, and - The step of mixing an extract with a predetermined amount of at least one nutritionally or pharmaceutically acceptable excipient to obtain an anti-hypercholesterolemia composition, The extract of the aforementioned Malabar spinach leaves is as follows: - A process of drying a predetermined amount of Malabar spinach leaves, and then grinding them to obtain ground plant material having a desired particle size. - A step of extracting ground plant material using a solvent selected from the group of ethanol, hexane, acetone, and redistilled water, followed by the removal and recovery of the solvent. - A step to obtain a crude extract by nitrogen flushing to remove residual solvent. - A step of dewaxing the crude extract by dissolving it in at least one of ethanol, methanol, and hexane, followed by deep freezing. - Prepared by cold filtering to remove unwanted waxy substances, followed by adding predetermined amounts of aerosil and dextrin to the crude extract to obtain an extract of Malabar spinach leaves. The Cordyceps sinensis powder mentioned above is as follows: - A step of growing a predetermined amount of Cordyceps culture in the form of mycelium or fruiting bodies, and It is prepared by the process of collecting the fruiting body or mycelium, drying it, and then pulverizing it to obtain Cordyceps sinensis powder. The extract of the aforementioned red yeast rice is as follows: - The process of grinding red yeast rice to obtain a powder. - A step of grinding or homogenizing with a solvent selected from the group consisting of water, ethanol, acetone, methanol, acetone, diethyl ether, and hexane, followed by filtration to obtain an extract. - A step of maintaining the extract for 8 to 12 hours to obtain a red precipitate and a yellow suspended solid. - A process to dry the yellow suspended material and obtain a slurry. - Prepared by adding a dried excipient to the slurry to obtain a powdered red yeast rice extract. A method for producing an anti-hypercholesterolemia composition.

11. - Extract of Malabar spinach leaves in the range of 30 to 70% by mass; - Red yeast rice extract in the range of 5 to 50% by mass; - Squalene extract in the range of 1 to 30% by mass; Cordyceps sinensis powder in the range of -10 to 35% by mass; and A composition for the treatment or prevention of atherosclerosis, comprising at least one nutritionally or pharmaceutically acceptable excipient in an amount ranging from 0.01 to 50% by mass.

12. The composition according to claim 11, wherein the at least one nutritionally or pharmaceutically acceptable excipient is selected from the group consisting of diluents, super-disintegrants, binders, lubricants, fillers, vitamins, minerals, vegetatives, and antioxidants.

Images