Antihypercholesterolemic 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 antihypercholesterolemic composition and a method for preparing the composition. [Background technology]

[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 needed for digestion. The cholesterol needed for these functions is either made by the body or obtained from various food sources, particularly animal sources such as egg yolks, meat, and cheese. Roughly speaking, cholesterol is divided into two categories: bad cholesterol (low-density lipoprotein - LDL) and good cholesterol (high-density lipoprotein - HDL). LDL deposits in blood vessels generally cause a reduction in vessel size, straining the flow of oxygen-rich blood throughout the body. Furthermore, LDL can also cause the formation of blood clots, which often break down and block blood flow, leading to heart attacks and strokes. HDL, on the other hand, transports cholesterol from other parts of the body to the liver for removal from the body.

[0003] Cholesterol is essential for the production of hormones, vitamin D, and substances needed for digestion, but higher levels of cholesterol can combine with other substances in the blood to form plaque. Over time, this plaque can adhere to the walls of arteries and cause coronary artery disease. Therefore, lowering cholesterol levels is of utmost importance. Hypercholesterolemia is elevated blood cholesterol levels, primarily due to cellular and plasma cholesterol concentrations. This leads to many heart diseases, such as atherosclerosis, coronary heart disease, myocardial infarction, and stroke.

[0004] There are many risk factors that can predispose people to having high cholesterol levels, such as unhealthy eating habits and lifestyle, lack of exercise, genetics, age, weight, and health conditions such as diabetes, smoking, gender, race, and ethnicity. Primarily, cholesterol is controlled either by medication or through dietary modification. Statins are generally the first-line medication for managing hypercholesterolemia. Although statins are first-line medications, they have a fairly poor safety record and high side effects, which can lead to a decline in long-term adherence to statins. Meanwhile, other cholesterol-lowering medications have a variety of side effects, including muscle pain and damage, liver damage, diabetes, confusion, and memory loss.

[0005] Therefore, there is a felt need for a composition that is free from the above-mentioned side effects and also has antihypercholesterolemic effects. Summary of the Invention

[0006] Accordingly, one aspect of the present invention provides a composition for reducing cholesterol formation, the antihypercholesterolemic composition comprising 30-70% by weight of an extract of Basalt alba leaf, 5-50% by weight of a red yeast rice extract, 1-30% by weight of a squalene extract, 10-70% by weight of a cordyceps extract, and 0.01-50% by weight of at least one nutritionally or pharmaceutically acceptable excipient.

[0007] Another aspect of the present invention also discloses a method for preparing the antihypercholesterolemic composition.

[0008] Reference will now be made to embodiments of the present invention, examples of which may be illustrated in the accompanying drawings, which are intended to be illustrative and not limiting, and while the invention will be described generally in the context of these implementations, it will be understood that it is not intended to limit the scope of the invention to these particular embodiments. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows histopathological images of the heart: 1 indicates nuclei, 2 indicates adipocytes, 3 indicates intercalated discs, 4 indicates 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 given with normal diet according to an embodiment of the present invention (OD). [Figure 2] Figure 2 shows histopathology of the spleen: 1 indicates trabeculae, 2 indicates white pulp, 3 indicates 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 given with normal diet (OD) according to an embodiment of the present invention. [Figure 3] FIG. 3 shows histopathological images of the lungs: 1 indicates bronchioles, 2 indicates alveoli, 3 indicates 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 given with normal diet according to an embodiment of the present invention (OD). [Figure 4] FIG. 4 shows histopathological images of kidneys: 1 indicates macula densa, 2 indicates proximal tubule, 3 indicates capillary, 4 indicates mesaglial cells, 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 given with normal diet according to an embodiment of the present invention (OD). [Figure 5]5 shows histopathological images of the liver: 1 indicates an artery, 2 indicates a bile duct, 3 indicates a portal duct, 4 indicates a 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. composition of the present invention given with normal diet (OD) according to an embodiment of the present invention. [Figure 6] FIG. 6 shows histopathological images of the aorta, where 1 indicates the intima, 2 indicates the media, 3 indicates the adventitia, and 4 indicates plaque, and A. normal rats, and B. HED diet rats showing intima changes with plaque formation in cholesterol-induced groups according to an embodiment of the present invention. [Figure 7] FIG. 7 shows histopathological images of the heart, where 1 indicates intercalated discs, 2 indicates nuclei, 3 indicates adipocytes, and 4 indicates myocardium, and 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), and G. composition of the present invention (BD) + high-fat diet (HFD) (continued) according to an embodiment of the present invention. [Figure 8] 8 shows histopathological images of the spleen: 1 indicates trabeculae, 2 indicates white pulp, 3 indicates central artery, 4 indicates red pulp, and 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), and G. composition of the present invention (BD) + high-fat diet (HFD) (continued) according to an embodiment of the present invention. [Figure 9] 9 shows histopathological images of the lungs: 1 indicates bronchioles, 2 indicates alveoli, 3 indicates veins, and 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 10] FIG. 10 shows histopathological images of the kidney: 1 indicates the macula densa, 2 indicates the proximal tubule, 3 indicates the capillary, and 4 indicates the mesaglial cells, and shows the results of 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] 11 shows histopathological images of the liver: 1 indicates an artery, 2 indicates a bile duct, 3 indicates a portal vein, 4 indicates a portal tract, and 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 12] FIG. 12 shows histopathological images of the aorta: 1 indicates plaque, 2 indicates intima, 3 indicates media, 4 indicates plaque degradation, 5 indicates plaque regeneration, and 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. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention discloses a composition for reducing cholesterol formation and a method for preparing the same. One aspect of the disclosed invention is an antihypercholesterolemic composition. The composition contains a cholesterol-lowering active ingredient 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 phytochemicals, or mixtures thereof, from the group consisting of Malabar Root, Red Yeast Rice, Cordyceps Sinensis, and squalene. These ingredients are obtained by conventional extraction procedures. In an exemplary embodiment of the invention, the cholesterol-lowering active ingredient is an extract, an enriched fraction, a pure compound, or a mixture thereof.

[0011] The extract of Malabar spinach is derived 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 is naturalized in China, tropical Africa, Brazil, Belize, Colombia, the West Indies, Fiji, and French Polynesia. Consequently, the extract or raw material can be obtained from any of these various natural sources. Generally, plant extracts are derived from plant material selected from various parts of the plant, including, but not limited to, rhizomes, roots, stems, seeds, bark, flowers, leaves, and fruits, and extracted using conventional extraction techniques with conventional solvents. In one embodiment of the present invention, the extract of Malabar spinach is derived from Malabar spinach leaves. 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 rotundifolia plants and products derived therefrom, but also extends to botanically closely related plants, particularly plants belonging to the same family, preferably the same genus, and more preferably plants belonging to the same species having substantially similar phenotypic and genotypic characteristics.

[0013] The conventional solvent may be selected from the group consisting of, but not limited to, water, alcohol, organic solvent and combinations thereof, or the method may be selected from the group, such as cold extraction, maceration, infusion, decoction, permeation, hot continuous extraction (Soxhlet), hydroalcoholic extraction, fermentation, countercurrent extraction, ultrasonic extraction (sonication), cold press extraction and supercritical fluid extraction, whichever is suitable to obtain a complete extract. The extract may be in solid or semi-solid or liquid form or in nanoemulsion form.

[0014] Red yeast rice, also known as red rice koji, red fermented rice, red koji rice, anka, or angkak, is produced by the fermentation of cooked rice grains with Monascaceae 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 have pigmentation capabilities that turn rice into reddish-purple grains. In one embodiment of the present invention, the monacolin K content in the red koji rice is 0.1 to 100 ppm, and the ankaflavin content in the red koji rice is 0.2 to 5%.

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

[0016] Cordyceps is also known as Chinese caterpillar fungus, Dong Chong Xia Cao, caterpillar mushroom, Cs-4, Champignon chenille, Chinese caterpillar fungus, Ophiocordyceps, Cordyceps sinensis, or vegetable caterpillar. Cordyceps is produced by a fermentation process, growing on cooked rice grains with Cordyceps cultures, preferably Cordyceps sinensis and Cordyceps militaris, which are grown as mat-like structures on rice grain substrates or in suspension by a fermentation process, producing mycelium, which in turn produces finger-like fruiting bodies resembling mushrooms. Cordyceps sinensis and Cordyceps militaris are native to Tibet, Nepal, and India. However, they are commonly spread throughout the Northern Hemisphere.

[0017] The scope of the present invention is not limited to Cordyceps sinensis fungi and products derived / extracted therefrom, but also extends to other closely related microorganisms, particularly those belonging to the same family, preferably the same genus, and more preferably those 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 olive, soybean, amaranth, rice sugar, grape seeds, almonds, coconut, palm, wheat germ, etc. Sources are not limited to plants but also include animal sources such as shark, marine sources, and animal origin. In one embodiment of the present invention, squalene extract is commercially available.

[0019] In one embodiment of the present invention, the amount of Malabar leaf extract is in the range of 30-70% by weight, the amount of red koji rice extract is in the range of 5-50% by weight, the amount of squalene extract is in the range of 1-30% by weight, the amount of Cordyceps sinensis extract is in the range of 10-70% by weight, and the amount of at least one nutritionally or pharmaceutically acceptable excipient is 0.01-50% by weight.

[0020] In another embodiment of the present invention, the amount of Malabar leaf extract is 35-55% by weight, the amount of red koji rice extract is 5-20% by weight, the amount of squalene extract is in the range of 1-5% by weight, the amount of Cordyceps sinensis extract is in the range of 15-35% by weight, and the amount of at least one nutritionally or pharmaceutically acceptable excipient is 5-20% by weight.

[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 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 invention, the 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, the at least one lubricant is selected from the group of corn starch and silicon dioxide.

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

[0026] In an embodiment of the present invention, the composition is prepared in a dosage form selected from the group consisting of semi-solid mass, oil and water-soluble dispersion, nanoemulsion, capsule, tablet, syrup, blend, suspension, etc. In another embodiment, the dosage form is further selected from the group consisting of immediate release, sustained release, and delayed release. In an exemplary embodiment of the present invention, the composition of the present invention is encapsulated and is in the form of a capsule. Depending on the dosage, the composition may further contain excipients necessary for the production of a more suitable dosage form and for disintegration after administration, 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, but not limited to, corn starch, gelatin, HPMC, and carrageenan.

[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 plaques, reducing the incidence of plaque formation and atherosclerosis. Alternatively, or additionally, the compositions may be for use in the management, treatment, or prevention of hypercholesterolemia.

[0029] In one embodiment of the present invention, an antihypercholesterolemic composition for treating or preventing hypercholesterolemia and atherosclerosis is disclosed, which comprises 30-70% by weight of Malus japonica leaf extract, 5-50% by weight of red koji rice extract, 1-30% by weight of squalene extract, 10-70% by weight of cordyceps sinensis extract, and 0.01-50% by weight of at least one nutritionally or pharmaceutically acceptable excipient.

[0030] In another aspect of the present invention, a method for preparing a composition is disclosed, which comprises first obtaining an extract of Malabar Malabar leaves, an extract of Cordyceps sinensis, an extract of Red Yeast Rice, and an extract of squalene, and then mixing predetermined amounts of these extracts with a predetermined amount of at least one nutritionally or pharmaceutically acceptable excipient to obtain the antihypercholesterolemic composition of the present invention.

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

[0032] In another embodiment of the present invention, red yeast rice extract is prepared by first 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, acetone, diethyl ether, and hexane. The resulting solvent extract is then filtered. Typically, the solvent extraction and filtration steps are repeated 2 to 5 times to obtain the maximum extract. The resulting extract is left for 8 to 12 hours to obtain a red precipitate and a yellow precipitate. The yellow precipitate is then dried in a rotary evaporator to obtain a slurry. A dry excipient is then added to the slurry to obtain a powdered red yeast rice extract having a monacolin K content ranging from 0.1 ppm to 100 ppm.

[0033] In yet another embodiment of the present invention, Monascus fungal extract is prepared by first growing a predetermined amount of Monascus broth on rice grains, followed by drying to remove moisture. The dried rice grains fermented with Monascus broth are then ground to obtain ground fungal material with the desired particle size suitable for maximum extraction. The Monascus broth powder is extracted with acetone, ether, methanol, ethanol, dichloromethane, ethyl acetate, chloroform, hexane, water, or a combination of solvents. This process is repeated 3 to 5 times to obtain the maximum extract. The extraction solvent is then evaporated to dryness using a vacuum dryer or evaporator. The concentrated extract is passed through a silica gel, Sephadex, or C18 column and eluted with an appropriate solvent or mixture thereof (acetone, ether, methanol, ethanol, dichloromethane, ethyl acetate, chloroform, hexane, or water) to recover the colored fraction. To further purify the colored fractions, repeated steps of column chromatography were employed using silica, Sephadex, or C18 column materials, and each fraction from the eluate was tested for monascin and ankaflavin content. Fractions rich in monascin and ankaflavin were collected, and further purification was carried out by preparative HPLC for high purity.

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

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

[0036] In one embodiment, the method of testing the purity of the ingredients and quantifying the active ingredients is performed using a spectrometer or gas chromatography or thin layer chromatography or high performance thin layer chromatography or high performance liquid chromatography or liquid chromatography or mass spectrometry. [Example]

[0037] Example 1 Comparison of the antiatherosclerotic and antihypercholesterolemic potential of the composition of the present invention with a statin (atorvastatin): Wistar albino rats weighing 190–225 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 day-night cycle. The experiments were conducted in accordance with the Japanese Association of Animal Ethics. (Prevention Research) A preventive study lasting 90 days used 6 rats per group. The study was conducted to determine the antihypercholesterolemic effects of a statin (atorvastatin) and the composition of the present invention. The 6 groups were as follows: 1. Normal control 2.High fat diet (HFD)lml 1 / d+normal diet 3. High-fat diet (HFD) + normal diet + composition of the present invention (OD) 4. High-fat diet (HFD) + normal diet + composition of the present invention (BD) 5. High-fat diet (HFD) + normal diet + statin 6. Composition of the present invention (OD) given with a normal diet A therapeutic study was conducted after 90 days of induction of cholesterol and atherosclerosis by HFD, and after 60 days of treatment, the effectiveness of the composition of the present invention was investigated in comparison with statins. Body weight was recorded to the nearest gram (g) by placing the subject on a balance at steady state and using an appropriate tare to 0.0000. The same weighing machine was used for all subjects. The machine was tested for several errors with a set of known 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 means and standard deviations of lipid profiles and hematological parameters, demonstrating that the composition of the present invention was able to maintain body weight even after HFD treatment, whereas statins were unable to. Furthermore, the reduction in total cholesterol was similar in both groups (the composition and the statin group) even after HFD treatment. However, significant inhibition was observed in total cholesterol synthesis (75%), LDL synthesis (85%), and triglycerides (50-70%). Apart from hemoglobin, which increased by 15% with twice-daily administration of the composition of the present invention, no significant changes were observed in blood parameters. (Biochemical analysis) Blood samples of 1.5-2 ml were collected from the retino-orbital chamber in vials for biochemical evaluation every 15 days. The investigations 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), WBC (10 3 / ul), RBC(10 6 / ul), Hb (g / dl), PCV (%), red blood cells (%), platelet count 10 5 / mm3 The analysis included the following: clotting time (seconds), neutrophils, lymphocytes, monocytes, and eosinophils. Evaluations were performed using the semi-automatic analyzer RX50 from MicroLabs Instruments, Erba Mannhelm, ERBA diagnostics Mannheml, Germany, and the Ecoline kit from COUNCELL21 from Tulip Diagnostics Pvt. Ltd. Lipid oxidation (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 kits.

[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). 1.0 ml of the filtrate was then treated with 0.5 ml of freshly prepared hydroxylamine reagent (alkaline hydroxylamine reagent for HMG-CoA) and mixed. After 5 minutes, 1.5 ml of iron chloride reagent was added to the same tube and shaken thoroughly. Measurements were taken at 540 nm after 10 minutes against a similarly treated saline / arsenate blank.

[0042] [Table 7]

[0043] The data presented in Table 7 show enzyme estimates after the animals were sacrificed. It was clear that HFD treatment induced hepatic lipid oxidation by 20%, which was significantly reduced by 23% by the group receiving the composition of the present invention twice daily and by 15% by statin treatment. Creatine phosphokinase activity was high in both HFDs, and the statin-treated groups (15-20%) showed muscle damage. The inhibition of SGOT (24%) and SGPT (29%) activity was greatest in the group receiving the composition of the present invention twice daily compared with statin (15% and 20%). Both the composition of the present invention and statin treatment maintained blood glucose levels similar to those of the normal diet group, even in the group receiving the HFD later. The inhibition of HMG-Co reductase activity by the composition of the present invention and statin was similar, i.e., 35%.

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

[0045] (Example 2) Histopathological analysis Figure 1 shows histopathological images of the heart. Histopathological findings of cardiac prevention studies showed significant fat deposition in the myocardium of HFD-induced rats compared with the hearts of normal rats. Meanwhile, in the case of statins, the myocardium showed myocardial relaxation as fat content decreased. However, rats treated with the composition 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 with all groups. Figure 2 shows the histopathological image of the spleen. The histopathological findings of the spleen in the prevention study showed that no significant changes were observed in any study group. However, this indicates the safety of the composition of the present invention in the spleen. Figure 3 shows the histopathological findings of the lungs. The histopathological findings of the lungs of various groups in the prevention study did not show any pathological changes in any study group. However, this indicates the safety of the composition of the present invention in the lungs. Figure 4 shows the histopathological image of kidney.The histopathological findings of the kidneys of various groups in the prevention study did not show any pathological changes in any study group.However, this indicates the safety of the composition of the present invention in kidney. Figure 5 shows histopathological images of the liver. Histopathological findings in the liver prevention study showed significant adipocyte (grade 2) formation in HFD-induced rats. However, intervention with the composition of the present invention at OD and BD dose levels showed a reduction in the rank of fatty liver from grade 2 to grade 1 (evidenced by dilation of the hepatic portal triad). In the statin group, dilation of the hepatic portal triad was more obvious 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 pads.

[0046] The experiment showed normal results, with normal weight gain. In the high-fat diet (HFD) 1 ml / d and normal diet groups, weight and lipid profile parameters showed significant increases compared to the normal control (P<0.005). No major changes were recorded between the high-fat diet (HFD) and normal diet and the composition of the present invention (OD), and the profiles were almost similar to those of the high-fat diet 1 ml / d and normal diet groups. However, the high-fat diet and 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, showing a significant correlation (P<0.005). Furthermore, the composition of the present invention (OD) given together with the normal diet also showed significant changes in biochemical profiling compared to the normal control group, and was found to be almost similar to the statin group. In multivariate analysis, the data suggested that the composition of the present invention (BD) showed improved effects compared to the statin group (P=0.070 and 0.058, respectively). Statistically, the data showed a significant correlation between biochemical and clinical profiles from day 0 to day 90 (P<0.001). Thus, the composition of the present invention exhibited a significant cholesterol-lowering effect even in the hypercholesterolemic Wistar rat model, i.e., continuous treatment with a high-fat diet, 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 during the entire study period and was found to be safe for animals in a house environment. Figure 7 shows histopathological images of the heart. Histopathological findings of the preventive study on the hearts of the group that continued to eat the HFD diet showed morphological changes (fat deposits) in the myocardium compared to the hearts of normal rats. Intervention using the composition of the present invention showed 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 deposits in the statin group, whereas significant improvement was evident in the group given the composition of the present invention.

[0047] Figure 8 shows the histopathological image of the spleen. The histopathological findings of the spleen prevention study in various groups showed no pathological changes in any of the study groups, thereby concluding that the composition of the present invention is safe in the spleen. Figure 9 shows the histopathological images of the lungs. The histopathological findings of the lung prevention study in various groups showed no pathological changes in any of the study groups, thereby concluding that the composition of the present invention is safe in the lungs. Figure 10 shows the histopathological images of the kidneys. The histopathological findings of the kidneys in various groups of the prevention study showed no pathological changes in any of the study groups, thereby concluding that the composition of the present invention is safe in the kidneys. Figure 11 shows histopathological images of the liver. Histopathological findings in the liver of the prevention study showed significant formation of adipocytes (grade 3) in HFD-induced rats (continuation), but intervention with statin and the composition of the present invention showed greater recovery in the group given the composition of the present invention (evidenced by dilation of the hepatic portal triad) compared to the statin group. Furthermore, the composition of the present invention improved the histopathology 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] From Tables 7 to 12, it can be inferred that the reductions in total cholesterol, LDL, and TG were significantly higher in the group using the composition of the present invention, i.e., 22%, 17%, and 26%, compared to 13%, 8%, and 11%, respectively, in the statin group. HDL levels also improved in the group using the composition of the present invention compared to the statin group, i.e., 44 mg / dl vs. 39 mg / dl.

[0051] [Table 15]

[0052] Table 11 clearly shows that the composition of the present invention reduced liver LPO by 18% compared to 11% for statins. The inhibition of HMG CO reductase activity by the composition of the present invention and statins was similar, i.e., 35%. SGOT reduction by the composition of the present invention was higher, i.e., 22%, compared to 16% for statins. Serum glutamic pyruvic transaminase (SGPT) reduction was significantly higher by the composition of the present invention, i.e., 23%, compared to statins, i.e., 5%.

[0053] (Example 4) Histopathological analysis of the aorta Figures 6 and 12 show the histopathological findings of rat aortas. Histopathological findings of plaques in the aortas after discontinuation of the high-fat diet demonstrated the successful development of an atherosclerotic rat model, and more severe plaque formation was observed in the HFD-continued group. Intervention with statin after discontinuation of the HFD showed a moderate reduction in plaques, while the statin-induced reduction with continued HFD was mild. The morphology of the group receiving the composition of the present invention showed complete plaque reduction / degradation, almost similar to that of normal rat aortas. Examples 1-4 show the most significant finding in the prevention study summarized above for the means and standard deviations 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 unable to. Furthermore, although the increase in TC was similar (10%) in both the composition of the present invention and statin groups, even after the HFD diet, HFD treatment clearly induced hepatic lipid peroxidation by 20%, which was significantly reduced (23%) by the group receiving the composition of the present invention (twice daily) and by 15% by statin treatment. Creatine phosphokinase activity was also elevated in both the HFD and statin-treated groups (8%-10%), which showed muscle damage, whereas no muscle damage was reported in the group receiving the composition of the present invention.

[0054] Furthermore, the inhibition of SGOT (24%) and SGPT (29%) activity was also found to be greatest 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 HFD-fed group.

[0055] Furthermore, histopathological findings from cardiac prevention studies showed significant fat deposition in the myocardium of HFD-induced rats compared with the hearts of normal rats. Meanwhile, in the case of statins, the myocardium showed myocardial relaxation as fat content decreased. Furthermore, rats treated with the composition of the present invention showed improved or better recovery of myocardial morphology, even during HFD treatment. Histopathological findings of the liver showed significant adipocyte formation (grade 2) in HFD-induced rats, but intervention with statins and the composition of the present invention at OD and OB dose levels resulted in a reduction in hepatic steatosis from grade 2 to grade 1 (evidenced by dilation of the hepatic portal triad). No significant changes were observed in all other groups. Statistically, the data showed a significant correlation between biochemical and clinical profiles 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 in a hypercholesterolemic Wistar rat model, even in the group fed a continuous high-fat diet, and showed improved results compared with statin (atorvastatin).

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

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

[0058] The present invention's composition at an oral 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 were correlated with histopathological analysis as follows: Histopathological findings of the liver showed significant formation of adipocytes (grade 3) in HFD-induced rats (continuation), but intervention with statin and the present invention's composition showed greater recovery (evidenced by dilation of the hepatic portal triad) in the group fed the present invention's composition compared to the statin group. Furthermore, the group fed the present invention's composition improved the pathology of fatty liver from grade 3 to grade 1. Histopathological findings of the aorta after discontinuation of the high-fat diet revealed plaque formation, indicating successful development of an atherosclerosis rat model, with more severe plaque formation observed in the HFD-continuation group. HFD discontinuation and statin intervention showed a moderate reduction in plaque, while the HFD-continuation and statin intervention showed only a mild reduction. The morphology of the group fed the present invention's composition showed complete plaque reduction / degradation, almost similar to that of normal rat aortas. Histopathological findings also showed the development of plaques after high-fat diet treatment, and the same plaques were observed to be completely removed / dissolved after treatment with the composition of the present invention.

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

[0060] Furthermore, the composition of the present invention reduced liver LPO by 18%, compared with 11% for statins. 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, 22%, compared with 16% for statins. The reduction in SGPT was significantly higher in the group receiving the composition of the present invention, i.e., 23%, compared with 5% for the statin group.

[0061] Furthermore, the examples also highlight liver damage / fatty liver associated with regular statin treatment, which correlates with histopathological analysis. CPK levels were found to increase by 10% in the statin group, reflecting muscle damage. The compositions of the present invention do not elevate CPK levels. Muscle damage is also associated with pain, such as muscle fatigue, caused by statin treatment. While sodium and potassium levels increased in the statin group, the compositions of the present invention were found to maintain Na (sodium) and K (potassium) levels. Long-term elevation of electrolytes, such as Na, is associated with blood pressure disorders that affect the heart. Thus, the compositions of the present invention are more effective than statins in terms of their anti-atherosclerotic and anti-hypercholesterolemic potential. In both preventive and therapeutic studies, oral doses of the compositions of the present invention at concentrations of 82.5 mg / kg body weight (OD&BD) and 135 mg / kg body weight (BD) showed no signs of toxicity or death and were thus found to be safe. Furthermore, histopathology and blood marker studies indicate that statin side effects such as vitamin D deficiency, liver damage, Na and K, and muscle fatigue are not associated with long-term use of the compositions of the present invention.

[0062] While the present invention has been described with respect to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made thereto without departing from the scope of the invention as defined in the claims.

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 ancaflavin 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 red yeast rice extract is as follows: - The process of grinding red yeast rice to obtain a powder. - A step of grinding or homogenizing 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 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.

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