Synergistic Hepatoprotective Composition

Abstract

To provide a synergic liver protective composition effective for protecting a normal liver function and having a novel combination of a plant chemical substance and carotenoid.SOLUTION: A liver protective composition according to one aspect includes curcuminoid and lutein with a ratio of 2:1 to 6:1. Regarding the particle size of the composition, D50 is in the range of 0.36 μm to 5 μm, and D90 is in the range of 0.60 μm to 10 μm. The curcuminoid and the lutein of the liver protective composition demonstrate synergic activity, and the curcuminoid and the lutein are present in the respective purified forms. The degree of purity of the curcuminoid is 80% to 95%, and the degree of purity of the lutein is 70% to 85%.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a synergistic hepatoprotective composition, particularly a synergistic hepatoprotective composition having a novel combination of phytochemicals, namely, curcuminoids and carotenoids such as lutein. The synergistic hepatoprotective composition is effective in promoting health, more particularly in protecting normal liver function. Also provided are novel methods for producing such compositions, as well as their use in foods, beverages including alcoholic beverages, and as nutraceuticals, dietary supplements, and pharmaceuticals. [Background technology]

[0002] Phytochemicals are biologically active compounds present in plants that are used in food and medicine. Phytochemicals such as curcuminoids and carotenoids are known for their health-promoting properties.

[0003] Turmeric (Curcuma longa) is a well-known Indian spice and a member of the ginger family (Zingiberaceae) widely cultivated for its rhizomes. Curcuminoids are polyphenolic compounds in turmeric rhizomes and are responsible for turmeric's yellow color. Turmeric's highly diverse pharmacological activities have also been reported. Curcumin is the major curcuminoid in turmeric. Two other curcuminoids are demethoxycurcumin and bis-demethoxycurcumin. Curcumin is one of the major components of turmeric involved in various biological actions. The biological effects of curcumin range from antioxidant and anti-inflammatory properties to inhibition of angiogenesis and have also been shown to have specific antitumor activity. Curcumin can bind heavy metals such as cadmium and lead, thereby reducing their toxicity. This property of curcumin explains its protective effect on the brain. Turmeric has been shown to have hepatoprotective properties similar to those of silymarin. The hepatoprotective effects of turmeric are primarily the result of its antioxidant properties as well as its ability to reduce the formation of proinflammatory cytokines (1).

[0004] Lutein, along with its isomer zeaxanthin and trace amounts of other carotenes such as beta-carotene and cryptoxanthin, generally belongs to a large class of plant pigments called carotenoids. Its presence in human tissues is entirely due to the ingestion of plant sources and is not synthesized by animal tissues. Lutein is present in a wide variety of fruits and vegetables (2) and imparts a yellow color to plants in which it is found, such as corn. Its concentrations are particularly high in leafy green vegetables such as spinach, collard greens, and kale (2). It is also present in some animal products, such as egg yolk, due to plant products eaten by animals (3). Various studies suggest that lutein may reduce the risk of developing cataracts and macular degeneration, two of the most common eye diseases in older adults. Oxidative stress is high in the eye due to intense light exposure and a high rate of oxidative metabolism in the retina. Lutein's antioxidant properties may reduce the extent to which oxidative damage promotes these diseases or minimize damage from oxidative stress by limiting the extent to which oxygen penetrates membranes (4, 5). Furthermore, associations have been found between individuals with the highest serum or dietary lutein concentrations and lower rates of coronary heart disease (6) or stroke (7). In two epidemiological studies, individuals with the highest serum concentrations of lutein + zeaxanthin had a significantly reduced risk of coronary heart disease, as measured by carotid artery thickness (8, 9). Lutein and zeaxanthin are transported into plasma largely by high-density lipoproteins (HDL) (10).

[0005] The liver is a vital organ that plays a key role in the metabolism and detoxification of various endogenous and exogenous harmful substances. The main causes of liver disease are toxic chemicals, excessive alcohol consumption, infectious diseases, and autoimmune diseases. It is in the liver that structural changes of drugs occur, resulting in biologically active or inactive metabolites, some of which are toxic. Therefore, the liver is a vulnerable target for damage by various chemicals and drugs.

[0006] Oxidative stress is considered a major contributing factor in the initiation and progression of liver damage. Various risk factors, such as alcohol and drugs, are known to induce oxidative stress, potentially leading to chronic diseases such as fatty liver disease (FLD). Fatty liver includes both alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD). Increasing evidence suggests a critical role for oxidative stress caused by the generation of reactive oxygen species (ROS) in the progression of ALD and NAFLD. Oxidative stress refers to the imbalance between the production of ROS and oxidants and the counteracting activity of antioxidants (11). Excessive ROS production adversely affects cellular function, ultimately contributing to fatty liver disease (FLD). In mitochondria, increased ROS production can cause mtDNA depletion, attack biomolecules (i.e., proteins, carbohydrates, and lipids), and damage mitochondrial membranes. It is noteworthy that mitochondria contain sufficient concentrations of phospholipids containing polyunsaturated fatty acids (PUFAs). PUFAs are more susceptible to oxidative damage due to the double bonds in their chemical structure, which leads to lipid peroxidation. PUFA peroxidation enhances the post-endoplasmic reticulum pre-secretory proteolysis of ApoB, thereby reducing VLDL secretion (12); this may further contribute to hepatic triglyceride (TG) accumulation. Furthermore, aldehydes formed by PUFA peroxidation impair cellular homeostasis because these molecules affect nucleotide and protein synthesis, reduce hepatic glutathione content, and increase production of the proinflammatory cytokine TNF-α (13). These effects lead to hepatocyte death and necrosis, inflammation, and liver fibrosis.

[0007] The liver is the primary site of alcohol metabolism. Liver damage or hepatotoxicity occurs through several interconnected pathways. The primary pathway for ethanol metabolism is the dehydrogenase system. Alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) cause the reduction of nicotinamide adenine dinucleotide (NAD) to NADH (the reduced form of NAD). An altered NAD / NADH ratio promotes hepatic steatosis through the inhibition of gluconeogenesis and fatty acid oxidation (14). Chronic alcohol exposure also activates hepatic macrophages, which produce tumor necrosis factor-alpha (TNF-α) (15). TNF-α induces mitochondria to increase the production of reactive oxygen species (ROS). This oxidative stress promotes hepatocellular necrosis and apoptosis.

[0008] Furthermore, as mentioned above, curcuminoids and lutein have been proven to be antioxidants and are useful for preventing diseases caused by oxidative stress. Therefore, it would be beneficial to prepare a composition containing curcuminoids and lutein, which has a potentially beneficial effect on liver health by protecting the liver from any oxidative damage, including damage caused by peroxidized polyunsaturated fatty acids.

[0009] Therefore, the object of the present invention is to provide a synergistic hepatoprotective composition comprising curcuminoids and lutein, which exhibits synergistic effects at very low dosages of both components and at the same time has high bioavailability, thereby effectively promoting liver health. Summary of the Invention

[0010] The present invention relates to a synergistic hepatoprotective composition, particularly a synergistic hepatoprotective composition having a novel combination of phytochemicals, namely, curcuminoids and carotenoids such as lutein. The synergistic hepatoprotective composition is effective in promoting health, more particularly in protecting normal liver health. Also provided are novel methods for producing such compositions, as well as their use in foods, beverages including alcoholic beverages, and as nutraceuticals, dietary supplements, and pharmaceuticals.

[0011] According to one embodiment of the present invention, a synergistic hepatoprotective composition is provided comprising curcuminoid and lutein.

[0012] According to one embodiment of the present invention, the synergistic hepatoprotective composition comprises curcuminoid and lutein, wherein the curcuminoid and lutein are present in a ratio ranging from 2:1 to 6:1.

[0013] According to another embodiment of the present invention, the ratio of curcuminoid to lutein ranges from 4:1 to 6:1 in the synergistic hepatoprotective composition.

[0014] According to another embodiment of the present invention, the synergistic hepatoprotective composition is effective in protecting the liver from liver damage or liver toxicity by attenuating the formation of ROS, balancing the NADH / NAD ratio, downregulating inflammatory pathways, and controlling hepatic steatosis.

[0015] According to another embodiment of the present invention, the synergistic hepatoprotective composition is effective in protecting the liver from liver damage or liver toxicity, which may be caused by various factors, such as unhealthy dietary patterns, high alcohol intake, intake of foods containing high amounts of peroxidized polyunsaturated fatty acids (PUFAs), or consumption of certain drugs.Furthermore, the hepatoprotective composition of the present invention is also effective in protecting the liver against non-alcoholic fatty liver disease caused by unhealthy dietary patterns.

[0016] According to another embodiment of the present invention, the synergistic hepatoprotective composition is effective in protecting the liver from hepatotoxicity by inhibiting oxidative stress damage in cells.

[0017] According to another embodiment of the present invention, the synergistic hepatoprotective composition is effective in protecting the liver from hepatic peroxidation, as investigated by the inventors through in vitro and in vivo studies to study the effectiveness of the synergistic hepatoprotective composition.

[0018] In accordance with yet another embodiment of the present invention, the synergistic hepatoprotective composition is effective in normalizing and maintaining lipid profiles, as well as maintaining healthy cholesterol levels and a healthy LDL / HDL ratio.

[0019] According to yet another embodiment of the present invention, the synergistic hepatoprotective composition is effective in protecting the liver from ethanol-induced toxicity, possibly through lipid-lowering and hepatoprotective activity.

[0020] According to another embodiment of the present invention, the synergistic hepatoprotective composition may be taken / administered periodically as a supplement or before, during or after the ingestion of fatty foods, drugs, and / or alcohol.

[0021] According to another embodiment of the present invention, there is provided a novel method for producing a synergistic hepatoprotective composition comprising curcuminoid and lutein, comprising: a) Curcuminoids and lutein are mixed in a predetermined ratio to form a dry blend; the particle size of the unprocessed dry blend is D 50 -20μm~50μm and D 90 -can be 100μm-200μm; b) adding suitable emulsifiers and maltodextrin to the dry blend; c) adding purified water to the dry blend to make a suspension; d) passing the suspension through a liquid colloid mill to form a uniform suspension; e) subjecting the suspension to further processing, such as passing it through a homogenizer or high shear particle wet mill, to produce a finely divided emulsion; f) further stirring the micronized emulsion at a speed of 25 rpm for 8-12 hours to bring the mass temperature to a temperature of 25°C-40°C, preferably 25°C-30°C; g) The micronized emulsion is subjected to further processing such as concentration and / or drying to obtain the synergistic hepatoprotective composition.

[0022] The method further comprises passing the dried synergistic hepatoprotective composition through a suitable particle sieve to obtain a uniform final product. The particle size of such processed compositions is D 50 -0.36μm to 5μm range, and D 90 can range from 0.60 μm to 10 μm. Such a reduction in particle size helps the synergistic hepatoprotective composition to have high bioavailability.

[0023] According to one embodiment of the present invention, the predetermined ratio of curcuminoid to lutein may range from 2:1 to 6:1.

[0024] According to another embodiment of the present invention, the predetermined ratio of curcuminoid to lutein may be in the range of 4:1 to 6:1.

[0025] In accordance with another embodiment of the present invention, the synergistic hepatoprotective composition may be added to food and / or beverage products or formulated into a dietary supplement.

[0026] According to another embodiment of the present invention, the synergistic hepatoprotective composition can be used as a dietary / nutritional supplement or as a therapeutic / health ingredient in various food and beverage products. Some examples of foods and beverages in which the synergistic hepatoprotective composition can be used as a health ingredient include, but are not limited to, tea, infusions, fruit juices, beverages, milk and dairy products, cereal-based products, alcoholic beverages, and processed foods.

[0027] According to another embodiment of the present invention, the synergistic hepatoprotective composition may also be formulated into a suitable dosage form selected from the group including powder, paste, tablet, syrup and / or capsule, and the like.

[0028] According to another embodiment of the present invention, the curcuminoid and lutein in the synergistic hepatoprotective composition are present in amounts much less than the recommended daily requirement and exhibit synergistic activity.

[0029] These and other features and aspects of the present invention are more clearly described in the complete specification.

[0030] BRIEF DESCRIPTION OF THE DRAWINGS The invention can be explained by the following drawings. [Brief explanation of the drawings]

[0031] [Figure 1(a)] FIG. 1(a) shows the cytotoxic effect of different concentrations of untreated curcuminoid (U1) on HepG2 cells. [Figure 1(b)] Figure 1(b) shows the cytotoxic effect of different concentrations of untreated lutein (U2) on HepG2 cells. [Figure 1(c)] FIG. 1(c) shows the cytotoxic effect of different concentrations of untreated synergistic hepatoprotective composition (U3) on HepG2 cells. [Figure 1(d)] Figure 1(d) shows the cytotoxic effect of different concentrations of treated curcuminoid (P1) on HepG2 cells. [Figure 1(e)] Figure 1(e) shows the cytotoxic effect of different concentrations of treated lutein (P2) on HepG2 cells. [Figure 1(f)] FIG. 1(f) shows the cytotoxic effect of different concentrations of the treated synergistic hepatoprotective composition (P3) on HepG2 cells. [Figure 2(a)] FIG. 2(a) shows the cytotoxic effect of different concentrations of untreated curcuminoid (U1) on ethanol-induced HepG2 cells. [Figure 2(b)]Figure 2(b) shows the cytotoxic effect of different concentrations of untreated lutein (U2) in ethanol-induced HepG2 cells. [Figure 2(c)] FIG. 2(c) shows the cytotoxic effect of different concentrations of untreated synergistic hepatoprotective composition (U3) on ethanol-induced HepG2 cells. [Figure 2(d)] Figure 2(d) shows the cytotoxic effect of different concentrations of treated curcuminoid (P1) on ethanol-induced HepG2 cells. [Figure 2(e)] Figure 2(e) shows the cytotoxic effect of different concentrations of treated lutein (P2) on ethanol-induced HepG2 cells. [Figure 2(f)] FIG. 2(f) shows the cytotoxic effect of different concentrations of the treated synergistic hepatoprotective composition (P3) on ethanol-induced HepG2 cells. [Figure 3(a)] FIG. 3(a) shows the hepatoprotective effects of untreated and treated synergistic hepatoprotective compositions on TBARS activity in ethanol-induced HepG2 cells. [Figure 3(b)] FIG. 3(b) shows the hepatoprotective effects of untreated and treated synergistic hepatoprotective compositions on SOD activity in ethanol-induced HepG2 cells. [Figure 3(c)] FIG. 3(c) shows the hepatoprotective effects of untreated and treated synergistic hepatoprotective compositions on GSH levels in ethanol-induced HepG2 cells. [Figure 4(a)] FIG. 4(a) is a bar graph showing the cytotoxic effect of the treated synergistic hepatoprotective composition on APAP-treated HepG2 cells. [Figure 4(b)] FIG. 4(b) is a bar graph showing the cytotoxic effect of silymarin in APAP-treated HepG2 cells. [Figure 5(a)] FIG. 5(a) shows a comparison of the hepatoprotective effects of the processed synergistic hepatoprotective composition with those of silymarin on TBARS activity in APAP-treated HepG2 cells. [Figure 5(b)]FIG. 5(b) shows a comparison of the hepatoprotective effect of the processed synergistic hepatoprotective composition with that of silymarin on SOD activity in APAP-treated HepG2 cells. [Figure 5(c)] Figure 5(c) shows a comparison of the hepatoprotective effects of the treated synergistic hepatoprotective composition with that of silymarin on GSH levels in APAP-treated HepG2 cells. [Figure 6] FIG. 6 shows the effects of the untreated and treated synergistic hepatoprotective compositions compared with the effect of silymarin on ethanol-induced intracellular ROS generation in HepG2 cells. [Figure 7] FIG. 7 shows a comparison of the effects of untreated and treated synergistic hepatoprotective compositions with the effect of silymarin on ethanol-mediated MMP reduction in HepG2 cells. [Figure 8] FIG. 8 shows a comparison of the effects of untreated and treated synergistic hepatoprotective compositions with the effect of silymarin on ethanol-induced nuclear apoptosis in HepG2 cells. [Figure 9] FIG. 9 shows a comparison of the effects of untreated and treated synergistic hepatoprotective compositions with the effect of silymarin on ethanol-induced nuclear fragmentation in HepG2 cells. [Figure 10] FIG. 10 shows a comparison of the effects of the processed synergistic hepatoprotective composition with that of silymarin on APAP-induced intracellular ROS generation in HepG2 cells. [Figure 11] FIG. 11 shows a comparison of the effects of the processed synergistic hepatoprotective composition with the effect of silymarin on APAP-mediated MMP reduction in HepG2 cells. [Figure 12] FIG. 12 shows a comparison of the effects of the processed synergistic hepatoprotective composition with the effect of silymarin on APAP-induced nuclear apoptosis in HepG2 cells. [Figure 13] FIG. 13 shows a comparison of the effects of the processed synergistic hepatoprotective composition with the effect of silymarin on APAP-induced nuclear fragmentation in HepG2 cells. [Figure 14]FIG. 14 shows the effect of the processed synergistic hepatoprotective composition and the effect of silymarin on ethanol-induced histopathological changes in the liver of rats on day 28 of the study. DETAILED DESCRIPTION OF THE INVENTION

[0032] The following describes several exemplary embodiments of the present invention. The invention, in its broader aspects, is not limited to the specific details and exemplary methods. Illustrative examples are described in this section with reference to the provided embodiments and methods. The invention according to its various aspects is particularly pointed out and distinctly claimed in the appended claims, read in light of this specification.

[0033] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a composition containing "a compound" includes a mixture of two or more compounds. It should also be noted that the term "or" is generally used in its sense including "and / or" unless the context clearly dictates otherwise.

[0034] The expression of various amounts by "%" means weight percent of the total solution or of the total component unless otherwise specified.

[0035] All cited references are incorporated herein by reference in their entirety. The citation of any reference does not constitute an admission of any determination regarding its availability as prior art to the claimed invention.

[0036] Unless otherwise defined, technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Reference is made herein to various methodologies and materials known to those skilled in the art.

[0037] Any suitable materials and / or methods known to those skilled in the art can be utilized in carrying out the present invention. However, preferred materials and methods are described. Materials, reagents, etc. referred to in the following description and examples are available from commercial sources unless otherwise noted.

[0038] The present invention is described in detail below in its product and process aspects.

[0039] The present invention relates to a synergistic hepatoprotective composition, particularly a synergistic hepatoprotective composition having a novel combination of phytochemicals, namely, curcuminoids and carotenoids such as lutein. The synergistic hepatoprotective composition is effective in promoting health, more particularly in protecting liver function. Also provided are methods for producing such compositions, as well as their use in foods, beverages, including alcoholic beverages and nutraceuticals, and / or as dietary supplements.

[0040] According to one embodiment of the present invention, a synergistic hepatoprotective composition is provided comprising curcuminoid and lutein.

[0041] According to one embodiment of the present invention, the synergistic hepatoprotective composition comprises curcuminoid and lutein in a ratio ranging from 2:1 to 6:1.

[0042] According to one embodiment of the present invention, the ratio of curcuminoid to lutein ranges from 4:1 to 6:1 in the synergistic hepatoprotective composition.

[0043] According to one embodiment of the present invention, the curcuminoids and lutein are present in their purified forms in the synergistic hepatoprotective composition, with the purity of the curcuminoids ranging from 80 to 95% and the purity of the lutein ranging from 70 to 85%.

[0044] According to one embodiment of the present invention, curcuminoids may be obtained / extracted and purified from the plant Curcuma longa, and lutein may be obtained / extracted and purified from the marigold plant Tagetes erecta or any other suitable plant source.

[0045] According to another embodiment of the present invention, the synergistic hepatoprotective composition is effective in protecting the liver from liver damage or liver toxicity.Some of the causative factors of liver damage or liver toxicity may be unhealthy eating patterns, excessive alcohol intake, certain medication intake, or the intake of foods containing a large amount of peroxidized polyunsaturated fatty acids (PUFA) in the diet.In addition, the synergistic hepatoprotective composition of the present invention is also effective in protecting the liver from non-alcoholic fatty liver disease caused by unhealthy eating patterns.

[0046] According to another embodiment of the present invention, the synergistic hepatoprotective composition is effective in protecting the liver from hepatotoxicity by its inhibitory effect on oxidative stress damage in cells.

[0047] According to another embodiment of the present invention, the synergistic hepatoprotective composition is effective in protecting the liver from oxidative stress damage in hepatocytes, which can be caused by various factors, such as alcohol consumption, drug consumption, or unhealthy dietary patterns, including the intake of high amounts of peroxidized PUFAs in the diet.

[0048] According to another embodiment of the present invention, the synergistic hepatoprotective composition is effective in protecting the liver from hepatic peroxidation induced by factors such as alcohol consumption, drug consumption, or unhealthy dietary patterns including the intake of high amounts of peroxidized PUFAs in the diet.

[0049] According to another embodiment of the present invention, as shown in in vitro studies, the synergistic hepatoprotective composition is effective in protecting the liver from hepatic peroxidation by reducing the production of the thiobarbituric acid reactive species (TBARS) malondialdehyde (MDA) in affected cells.

[0050] According to another embodiment of the present invention, the synergistic hepatoprotective composition is effective in protecting the liver from hepatotoxicity by increasing the levels of the non-enzymatic antioxidants glutathione reductase (GSH) and superoxide dismutase (SOD) in affected cells. A decrease in non-enzymatic antioxidants can be caused by various factors, such as alcohol consumption, drug intake, or unhealthy dietary patterns, including the intake of large amounts of peroxidized PUFAs in the diet.

[0051] According to another embodiment of the present invention, the synergistic hepatoprotective composition is effective in protecting the liver from liver damage or liver toxicity by attenuating the formation of ROS, balancing the NADH / NAD ratio, downregulating inflammatory pathways, and controlling hepatic steatosis.

[0052] According to another embodiment of the present invention, the synergistic hepatoprotective composition of the present invention protects the liver from hepatotoxicity by preventing reactive oxygen species (ROS) generation in affected cells, thereby effectively attenuating further degenerative pathways of inflammation in affected cells. Increased ROS generation can be caused by various factors, such as alcohol consumption, drug consumption, or unhealthy dietary patterns, including the intake of large amounts of peroxidized PUFAs in the diet. The synergistic hepatoprotective composition also effectively protects the liver against hepatotoxicity induced by factors such as alcohol, drugs, and unhealthy dietary patterns by preventing depolarization of the mitochondrial membrane in affected cells.

[0053] In one embodiment of the present invention, as established by in vitro studies, the synergistic hepatoprotective composition of the present invention effectively protects the liver from hepatotoxicity induced by factors such as alcohol, drugs, and unhealthy dietary patterns by preventing cellular nuclear apoptosis and nuclear fragmentation in affected cells.

[0054] In yet another embodiment of the present invention, as established by preclinical studies, the synergistic hepatoprotective composition of the present invention is effective in suppressing increased levels of serum enzymes, namely alanine aminotransferase (ALT), aspartate aminotransferase (AST), alcohol dehydrogenase (ADH) and gamma glutamyltransferase (GGT).

[0055] According to another embodiment of the present invention, the synergistic hepatoprotective composition is effective in protecting the liver from hepatic peroxidation, as investigated by the inventors through in vitro and in vivo studies to study the effectiveness of the synergistic hepatoprotective composition.

[0056] According to yet another embodiment of the present invention, the synergistic hepatoprotective composition is effective in normalizing and maintaining lipid profiles, as well as maintaining healthy cholesterol levels and a healthy LDL / HDL ratio.

[0057] According to yet another embodiment of the present invention, the synergistic hepatoprotective composition is effective in protecting the liver from hepatotoxicity induced by factors such as alcohol, drugs, and unhealthy dietary patterns, possibly through lipid-lowering and hepatoprotective activity.

[0058] According to another embodiment of the present invention, the curcuminoids and lutein are obtained from natural sources that are known to have no side effects.

[0059] According to another embodiment of the present invention, the synergistic hepatoprotective composition may be taken / administered periodically as a supplement or before, during or after the ingestion of fatty foods, drugs and / or alcohol.

[0060] The present invention also provides a method for producing a synergistic hepatoprotective composition comprising lutein and curcuminoid, comprising the steps of: a) Curcuminoids and lutein are mixed in a predetermined ratio to form a dry blend; the particle size of the unprocessed dry blend is D 50-20μm~50μm and D 90 -can be 100μm~200μm; b) adding suitable emulsifiers and maltodextrin to the dry blend; c) adding purified water to the dry blend to make a suspension; d) passing the suspension through a liquid colloid mill to form a uniform suspension; e) subjecting the suspension to further processing, such as passing it through a homogenizer or high shear particle wet mill, to produce a finely divided emulsion; f) further stirring the micronized emulsion at a speed of 25 rpm for 8-12 hours to bring the mass temperature to a temperature of 25°C-40°C, preferably 25°C-30°C; g) The micronized emulsion is subjected to further processing such as concentration and / or drying to obtain the synergistic hepatoprotective composition.

[0061] In the above method, the curcuminoids incorporated for preparing the synergistic hepatoprotective composition may be 80-95% pure, and the purity of lutein may range from 70-85%.Furthermore, some examples of emulsifiers that may be used include, but are not limited to, modified starch, gum ghatti, gum arabic, gum acacia, methylcellulose, and sucrose fatty acid esters.

[0062] The method further includes passing the dried hepatoprotective composition through a suitable particle sieve to obtain a uniform final product. The particle size of the processed composition is D 50 -0.36μm to 5μm, D 90 The particle size can range from 0.60 μm to 10 μm. Such a reduction in particle size helps the synergistic hepatoprotective composition to have high bioavailability.

[0063] According to one embodiment of the present invention, the predetermined ratio of curcuminoid to lutein may range from 2:1 to 6:1.

[0064] According to another embodiment of the present invention, the predetermined ratio of curcuminoid to lutein may be in the range of 4:1 to 6:1.

[0065] According to another embodiment of the present invention, the synergistic hepatoprotective composition may be added to food and / or beverage products, formulated into dietary supplements, nutraceuticals and / or pharmaceuticals.

[0066] According to another embodiment of the present invention, the synergistic hepatoprotective composition can be used as a dietary supplement, a pharmaceutical, a dietary / nutraceutical, or as a therapeutic / health ingredient in various food and beverage products. Some examples of foods and beverages in which the synergistic hepatoprotective composition can be used as a health ingredient include, but are not limited to, tea, infusions, fruit juices, beverages, milk and dairy products, cereal-based products, alcoholic beverages, and processed foods.

[0067] According to another embodiment of the present invention, the synergistic hepatoprotective composition may also be formulated into a suitable dosage form selected from the group including powder, paste, tablet, syrup, infusion and / or capsule, etc.

[0068] According to another embodiment of the present invention, the curcuminoid and lutein of the synergistic hepatoprotective composition are present in amounts much less than the recommended daily requirement and exhibit synergistic activity.

[0069] In one embodiment of the present invention, the synergistic hepatoprotective composition may also be added to any alcoholic beverage to counteract the adverse effects of alcohol while it is being consumed by a person.

[0070] The following examples are intended to further illustrate certain preferred embodiments of the invention and are not limiting in nature. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. [Example]

[0071] Example 1 This example describes representative synergistic hepatoprotective compositions of the present invention containing curcuminoids and lutein in different weight ratios. Table 1 shows the components and their amounts used in the synergistic hepatoprotective compositions with different ratios of curcuminoids and lutein.

[0072] [Table 1]

[0073] Example 2 A synergistic hepatoprotective composition as detailed in Example 1 was prepared by the method described below: Curcuminoids (95% purity) and lutein (80% purity) were mixed with modified starch emulsifier and maltodextrin in predetermined amounts to prepare a dry blend (100 g). The particle size of the dry blend was D 50 -20 μm, D 90 The particle size was -100 μm. This dry blend was then reconstituted with 333.4 mL of RO purified water to form a suspension, which was further stirred until no lumps were present in the suspension. The suspension was then pre-emulsified in a liquid colloid mill or liquid wet mill to form a uniform emulsion. The resulting pre-emulsion was passed through a high-pressure homogenizer / high-shear particle wet mill to generate submicron-sized emulsion droplets. The micronized emulsion was further stirred at a speed of up to 25 rpm for 8-12 hours to achieve a mass temperature of 25°C-40°C, preferably 25°C-30°C. This micronized emulsion was further spray-dried at a chamber temperature of 110°C or less. The spray-dried coarse powder was then collected and subjected to dry milling using either a micropulverizer or air-assisted high-shear dry milling. The milled particles were collected, passed through an appropriate particle sieve, and mixed to obtain the final product. The particle size of the final powder was D 50 -0.360 μm, D 90 It was -0.60 μm.

[0074] Example 3 A synergistic hepatoprotective composition as detailed in Example 1 was prepared by the method described below: Curcuminoids (90% pure) and lutein (75% pure) were mixed with modified starch emulsifier and maltodextrin in predetermined amounts to prepare a dry blend (100 g). The particle size of the dry blend was D 50 -40 μm, D 90 The particle size was -155 μm. This dry blend was then reconstituted with 333.4 mL of RO purified water to form a suspension, which was further stirred until no lumps were present in the suspension. The suspension was then pre-emulsified in a liquid colloid mill or liquid wet mill to form a uniform emulsion. The resulting pre-emulsion was passed through a high-pressure homogenizer / high-shear particle wet mill to generate submicron-sized emulsion droplets. The micronized emulsion was further stirred at a speed of up to 25 rpm for 8-12 hours to achieve a mass temperature of 25°C-40°C, preferably 25°C-30°C. This micronized emulsion was further spray-dried at a chamber temperature of 110°C or less. The spray-dried coarse powder was then collected and subjected to dry milling using either a micropulverizer or air-assisted high-shear dry milling. The milled particles were collected, passed through an appropriate particle sieve, and mixed to obtain the final product. The particle size of the final powder was D 50 -2.76 μm, D 90 -5.44 μm.

[0075] Example 4 A synergistic hepatoprotective composition as detailed in Example 1 was prepared by the method described below: Curcuminoids (85% purity) and lutein (70% purity) were mixed with modified starch emulsifier and maltodextrin in predetermined amounts to prepare a dry blend (100 g). The particle size of the dry blend was D 50 -50μm,D 90The particle size was -200 μm. This dry blend was then reconstituted with 333.4 mL of RO purified water to form a suspension, which was further stirred until no lumps were present in the suspension. The suspension was then pre-emulsified in a liquid colloid mill or liquid wet mill to form a uniform emulsion. The resulting pre-emulsion was passed through a high-pressure homogenizer / high-shear particle wet mill to generate submicron-sized emulsion droplets. The micronized emulsion was further stirred at a speed of up to 25 rpm for 8-12 hours to achieve a mass temperature of 25°C-40°C, preferably 25°C-30°C. This micronized emulsion was further spray-dried at a chamber temperature of 110°C or less. The spray-dried coarse powder was then collected and subjected to dry milling using either a micropulverizer or air-assisted high-shear dry milling. The milled particles were collected, passed through an appropriate particle sieve, and mixed to obtain the final product. The particle size of the final powder was determined by the D 50 -5 μm, D 90 -10 μm.

[0076] Example 5 In vitro studies to evaluate the hepatoprotective effects of the synergistic hepatoprotective composition against ethanol-induced hepatotoxicity and drug-induced hepatotoxicity the purpose: The main purpose of this study was to investigate the effects of the synergistic hepatoprotective composition of the present invention and silymarin on ethanol and acetaminophen (APAP)-induced hepatotoxicity by suppressing oxidative stress damage in HepG2 cell line. Silymarin is a widely used hepatoprotective agent, so it was used to compare the effects of the novel synergistic hepatoprotective composition of the present invention.

[0077] Cell lines and media: This experiment was performed on human hepatoma (HepG2) cells. HepG2 cells were obtained from the National Centre for Cell Science, Pune, India. Cells were maintained in DMEM medium containing 10% FBS, 1% glutamine, and 100 U penicillin-streptomycin at 37°C in a 5% CO atmosphere. Stocks were stored in T-75cm 2 Maintained in tissue culture flasks.

[0078] experiment Cytotoxicity test The non-toxic concentrations of unprocessed curcuminoids (U1), unprocessed lutein (U2), unprocessed synergistic hepatoprotective composition (U3), and processed curcuminoids (P1), processed lutein (P2) and processed synergistic hepatoprotective composition (P3), silymarin, and its preventive effects against ethanol- and acetaminophen (APAP)-induced cytotoxicity were evaluated by MTT assay.

[0079] Sample preparation: 1. The raw curcuminoid (U1) sample was prepared by taking purified curcuminoid and mixing it with the dry ingredients, i.e., emulsifier and maltodextrin, in a predetermined amount. 2. Unprocessed lutein (U2) sample was prepared by taking purified lutein and mixing it with the dry ingredients, i.e., emulsifier and maltodextrin, in a predetermined amount. 3. The unprocessed synergistic hepatoprotective composition (U3) sample was prepared by mixing curcuminoids and lutein in a predetermined ratio and then dry-mixing the mixture with emulsifiers and maltodextrin in a predetermined amount. 3. Processed curcuminoid (P1) sample was prepared by taking purified curcuminoid and mixing it with dry ingredients, i.e., emulsifier and maltodextrin. This dry blend was then subjected to the same process as described for the synergistic hepatoprotective composition in any of Examples 2-4 above. 4. Processed lutein (P2) samples were prepared by taking purified lutein and mixing it with the dry ingredients, i.e., emulsifier and maltodextrin. This dry blend was then subjected to the same treatment as described for the synergistic hepatoprotective composition in any of Examples 2-4 above. 5. Treatment A synergistic composition sample (P3) was prepared as described in any of Examples 2-4 above.

[0080] HepG2 cells were harvested and plated in a 96-well plate at a concentration of 1 × 10 5 Cells were seeded at 1000 μg / well (1.95, 3.90, 7.81, 15.62, 31.25, 62.50, 125, 250, 500, and 1000 μg / mL). To measure the toxicity of U1, U2, U3, P1, P2, and P3 (1.95, 3.90, 7.81, 15.62, 31.25, 62.50, and 125 μg / mL) for 24 h, an MTT assay was performed. To evaluate the therapeutic effects of U1, U2, U3, P1, P2, and P3 against ethanol and APAP, cells were pretreated with different concentrations of U1, U2, U3, P1, P2, and P3 (3.90, 7.81, 15.62, 31.25, 62.50, and 125 μg / mL) and silymarin (3.12–200 μg) 1 h before exposure to ethanol (100 mM) and acetaminophen (APAP) (20 mM). Then, 100 μL of MTT solution (5 mg / mL in PBS) was added and the incubation was extended for another 4 hours. Then, 100 μL of DMSO was added and the absorbance was measured at 570 nm using a multimode plate reader.

[0081] Lipid peroxidation and antioxidant enzyme activity assays The concentration of malondialdehyde (MDA), an indicator of lipid peroxidation, was determined based on thiobarbituric acid reactive species (TBARS) production, superoxide dismutase (SOD) activity, and glutathione reductase (GSH) activity measured according to kit methods (Himedia and Sigma). Samples were measured by standard spectrophotometric methods.

[0082] Cells were incubated with ethanol (100 mM), U1, U2, U3, P1, P2, and P3, and silymarin for 24 hours. Total GSH content, SOD activity, and lipid peroxidation in the hepatoprotective compositions were measured.

[0083] Cells were incubated with APAP (20 mM) and P3, silymarin, for 24 hours. Total GSH content, SOD activity, and lipid peroxidation in the hepatoprotective composition were measured.

[0084] Measurement of intracellular ROS ROS was measured using a non-fluorescent probe, 2,7-diacetyldichlorofluorescein (DCFH-DH), which can penetrate the intracellular matrix of cells, where it is oxidized by ROS to fluorescent dichlorofluorescein (DCF). Briefly, an aliquot (8 × 10) of isolated cells was cultured in a 5% CO2-free well. 6 The cells / mL were prepared in normal phosphate-buffered saline (pH 7.4) to a final volume of 2 mL, and 1 mL of cell aliquots were taken, to which 1 μL of DCFH-DA (1 mg / mL) was added and incubated for 30 min at 37°C under dark conditions. Images were taken with an epifluorescence microscope (Nikon Eclipse TS100, Japan) equipped with a digital camera (Nikon Coolpix 4500, Japan).

[0085] HepG2 cells were treated with 62.50 μg of U1, U2, U3, P1, P2, or P3 and 50 μg of silymarin for 1 h, followed by 100 mM ethanol for 24 h. Intracellular ROS accumulation was measured using the fluorescent probe DCF-DA.

[0086] Measurement of mitochondrial membrane potential A decrease in MMP is a sign of early apoptosis. In this study, differences in MMP were detected using the fluorescent dye rhodamine 123 (Rh123). HepG2 cells were cultured in 6-well plates (1 × 10 6) and harvested after the treatments described above. After 24 hours of incubation with the test compound and ethanol or APAP, the cells were incubated with Rh123 (5 mmol / mL) for 15 minutes. The cells were then rinsed with PBS, and fluorescence was observed under a fluorescence microscope using a blue filter (450-490 nm).

[0087] Dual fluorescence and DAPI staining of HepG2 cells HepG2 cells were grown in 35 mm cell culture plates and treated with each candidate compound P3 (62.50 μg), silymarin (50 μg), and 100 mM ethanol or APAP (20 mM) for 24 hours, then washed with Dulbecco's phosphate-buffered saline (DPBS). Morphological changes associated with ethanol- or APAP-induced hepatotoxicity were detected using acridine orange / ethidium bromide (AO / EB) fluorescent staining. Ethidium bromide (100 mg / mL) and acridine orange (100 mg / mL) were mixed 1:1, and 100 mg / mL of DAPI was added to the cells. Morphological characteristics were subsequently evaluated by fluorescence microscopy.

[0088] result: A. Effects of untreated and treated components and synergistic hepatoprotective compositions on HepG2 cells Figure 1(a, b, c) shows the cytotoxic effects of different concentrations of the untreated components and the synergistic hepatoprotective composition on HepG2 cells. Figure 1(d, e, and f) shows the cytotoxic effects of different concentrations of the treated components and the synergistic hepatoprotective composition on HepG2 cells.

[0089] Figure 2(a, b, c) shows the cytotoxic effects of different concentrations of processed ingredients and synergistic hepatoprotective composition on ethanol-induced HepG2 cells, while Figure 2(d, e, f) shows the cytotoxic effects of different concentrations of processed ingredients and synergistic hepatoprotective composition on ethanol-induced HepG2 cells.

[0090] MTT assays showed that cell viability remained unchanged in HepG2 cells at low concentrations of 1.95–62.50 μg / mL. Furthermore, concentrations of U1, U2, U3, P1, P2, and P3 at 3.9–62.50 μg / mL resulted in a significant increase in cell viability compared to ethanol. The maximum cell viability was observed when exposed to 62.50 μg / mL (Figure 2(a, b, c, d, e, and f)).

[0091] Therefore, 62.50 μg / mL concentration of U1, U2, U3, P1, P2, P3 was selected as the optimal protective concentration for all further studies.

[0092] B. Hepatoprotective Effects of Untreated and Treated Components and Synergistic Hepatoprotective Compositions on Antioxidant Enzyme Activities in Ethanol-Induced HepG2 Cells A TBARS assay was performed to study the ability of U1, U2, U3, P1, P2, P3, and silymarin to alter ethanol-induced lipid peroxidation in HepG2 cells (Figure 3a). The results showed higher TBARS formation in the ethanol-stimulated group compared to the control cells. However, TBARS formation was reduced in the treated cells, respectively.

[0093] The effects of U1, U2, U3, P1, P2, P3, and silymarin on ethanol-induced activity of SOD and GSH levels are shown in Figures 3(b) and 3(c). Ethanol stimulation significantly reduced the levels of the nonenzymatic antioxidant GSH and the activity of SOD in HepG2 cells compared with control cells. Interestingly, SOD and GSH increased in U1, U2, U3, P1, P2, P3, and silymarin-treated cells compared with the ethanol-stimulated group.

[0094] Furthermore, the effects of the treated synergistic hepatoprotective composition (P3) were comparable to those of silymarin and the control group, and were better than those of the untreated composition and the individual components curcuminoids (U1, P1) and lutein (U2, P2).

[0095] Therefore, from these results it was inferred that the processed form of the composition of the present invention (P3) exhibits synergistic activity of the individual components (i.e., curcuminoids and lutein).

[0096] C. Cytotoxic Effects of Treated Synergistic Hepatoprotective Composition vs. Silymarin in Acetaminophen (APAP)-Treated HepG2 Cells 4(a) and 4(b) show a comparison of the cytotoxic effects of the treated synergistic hepatoprotective composition (P3) versus silymarin in APAP-treated HepG2 cells.

[0097] In this study, APAP exposure caused significant cytotoxicity in HepG2 cells. However, pretreatment with the synergistic hepatoprotective composition (P3) (62.50 μg) significantly suppressed APAP-induced cytotoxicity in HepG2 cells; the results were comparable to those of silymarin (50 μg).

[0098] D. Hepatoprotective Effects of the Treated Synergistic Hepatoprotective Composition Versus Silymarin on Antioxidant Enzyme Activity in Acetaminophen (APAP)-Induced HepG2 Cells From Figure 5(a), it can be inferred that the levels of TBARS increased in APAP-induced HepG2 cells compared to the control treatment. Furthermore, treatment with 62.50 μg of the administered synergistic hepatoprotective composition (P3) prior to APAP induction significantly suppressed APAP-mediated TBARS levels. These results were comparable to the effects exhibited by silymarin (50 μg).

[0099] Antioxidants act as a primary defense against free radicals. APAP-induced HepG2 cells significantly reduced cellular antioxidant status due to excessive ROS generation. Conversely, treatment with 62.50 μg of the processed synergistic hepatoprotective composition (P3) and 50 μg of silymarin given before APAP induction significantly reduced APAP-induced loss of antioxidant status (SOD and GSH levels) in HepG2 cells, as seen in Figures 5(b) and (c).

[0100] E. Effects of untreated and treated synergistic hepatoprotective composition vs. silymarin on ROS generation in ethanol-induced cells From Figure 6, it can be inferred that ROS levels increased in HepG2 cells stimulated with ethanol alone compared with the untreated control group. Conversely, U1, U2, U3, P1, P2, P3 (62.50 μg / mL), and silymarin (50 μg / mL) significantly suppressed ethanol-induced ROS generation in HepG2 cells. The reduction in ROS generation by treatment with 62.50 μg / mL of the synergistic hepatoprotective composition (P3) was comparable to that by treatment with 50 μg / mL of silymarin.

[0101] F. Effect of untreated and treated synergistic hepatoprotective composition vs. silymarin on ethanol-mediated MMP reduction Figure 7 shows that mitochondrial membrane potential was significantly reduced in cells exposed to 100 mM ethanol for 24 hours compared to controls. Depolarization of the mitochondrial membrane potential induced by outer membrane damage resulted in loss of dye from mitochondria and reduced intracellular fluorescence compared to controls. Conversely, 24-hour pretreatment with U1, U2, U3, P1, P2, and P3 at 62.50 μg / mL and silymarin at 50 μg / mL attenuated the ethanol-induced mitochondrial membrane depolarization, as evidenced by increased fluorescence intensity.

[0102] The results showed that the processed synergistic hepatoprotective composition (P3) could significantly increase intracellular fluorescence, thereby indicating a positive effect in preventing the depolarization of cellular mitochondrial membrane potential, and these results were comparable to the effect of the known hepatoprotective agent silymarin.

[0103] G. Effect of untreated and treated synergistic hepatoprotective composition vs. silymarin on ethanol-induced nuclear apoptosis Figure 8 shows the fluorescence microscopic morphological changes in control and ethanol-induced HepG2 cells after staining with AO / EtBr. The figure shows micrographs showing the effect of 62.50 μg of the treated synergistic hepatoprotective composition (P3) and 50 μg of silymarin on the apoptotic morphological changes induced by 100 mM ethanol in HepG2 cells. It can be seen that the treated synergistic hepatoprotective composition (P3) inhibited apoptosis, as shown by AO / EtBr staining in HepG2 cells, which was comparable to the effect exhibited by silymarin.

[0104] H. Effect of Untreated and Treated Synergistic Hepatoprotective Composition vs. Silymarin on Ethanol-Induced Nuclear Fragmentation Treatment of HepG2 cells with 100 mM ethanol caused nuclear shrinkage and fragmentation, as shown in Figure 9. It can be seen that both the treated synergistic hepatoprotective composition (P3) and silymarin treatment before ethanol exposure reduced nuclear shrinkage and fragmentation, which may be due to their strong radical scavenging properties.

[0105] I. Effects of the Synergistic Hepatoprotective Composition vs. Silymarin on APAP-Induced Intracellular ROS Generation Treatment of HepG2 cells with 20 mM APAP caused an increase in DCF fluorescence. Pretreatment of cells with the synergistic hepatoprotective composition (P3) at 62.50 μg / mL reduced DCF fluorescence, indicating a significant reduction in APAP-induced free radical release compared with the group treated with APAP alone (FIG. 10).

[0106] J. Effect of processed synergistic hepatoprotective composition vs. silymarin on acetaminophen (APAP)-mediated MMP reduction As shown in Figure 11, mitochondrial membrane potential (ΔΓm) was measured by measuring the green fluorescence ratio after Rh-123 treatment. APAP-treated cells were polarized and exhibited a large ΔΓm, with reduced green fluorescence compared to the control. Pretreatment of cells with the treated synergistic hepatoprotective composition (P3) increased green fluorescence, indicating a depolarized state of the mitochondrial membrane, compared to cells treated with APAP alone. Furthermore, the treated synergistic hepatoprotective composition (P3) and silymarin each exhibited similar effects.

[0107] K. Effect of the Synergistic Hepatoprotective Composition vs. Silymarin on APAP-Induced Nuclear Apoptosis Figure 12 shows micrographs showing the effects of the processed synergistic hepatoprotective composition (P3) at 62.50 μg and 50 μg of silymarin on 20 mM APAP-induced apoptotic morphological changes in HepG2 cells. From Figure 12, it can be inferred that the processed synergistic hepatoprotective composition (P3) inhibited apoptosis as stained by AO / EtBr staining in HepG2 cells, and the results were comparable to those shown by silymarin.

[0108] L. Effect of the Synergistic Hepatoprotective Composition vs. Silymarin on APAP-Induced Nuclear Fragmentation Treatment of HepG2 cells with 20 mM APAP caused nuclear shrinkage and fragmentation, and morphological changes were observed in the cells, as seen in Figure 13. Pretreatment with the synergistic hepatoprotective composition (P3) and silymarin before APAP exposure suppressed nuclear shrinkage and fragmentation compared to cells treated with APAP alone.

[0109] Example 6 - In Vivo Testing Hepatoprotective effect of synergistic hepatoprotective composition against alcohol-induced liver injury in Wistar rats Test Objective: 1. To monitor the effect of synergistic hepatoprotective composition (P3) on alcohol-induced liver injury changes in the initial and final body weight of experimental rats 2. To evaluate the effect of synergistic hepatoprotective composition (P3) at different doses on alcohol-induced hepatotoxicity by assessing lipid profile levels TG, TCH, LDL, and HDL 3. Measuring the activity of liver marker enzymes AST, ALT, ADH and GGT 4. To evaluate the effect of Synergistic Hepatoprotective Composition (P3) at different doses on alcohol-induced hepatotoxicity by assessing lipid peroxidation markers MDA and 4-HNE. 5. To assess inflammatory (TNF-α, IL-6 and IL-1) markers during alcohol-induced hepatotoxicity in rats.

[0110] material and method: animal- Male Wistar rats weighing 130–150 g were obtained from Biogen Laboratory Animal Facility (India). o Animals were acclimatized in conventional vivariums at 25°C with a 12-h light:dark cycle. Animals were fed standard rat chow and water ad libitum. Food was withdrawn 18–24 h before the experiment. The care and use of experimental animals was carried out in accordance with the guidelines of the Council Directive CPCSEA, India on Good Laboratory Practice for Animal Experiments (MCAS / IAEC / 2019 / 6 / 9 / 2 / 2019).

[0111] Experimental design and treatment plan: Experimental male Wistar rats were divided into six groups with six animals in each group and analyzed over a total experimental period of 28, and the dosages of ethanol, silymarin and processed synergistic hepatoprotective composition P3 were as follows: Group 1: Control (vehicle) Group 2: ethanol (EtOH) in 0.9% saline (2.4 mg / kg body weight) Group 3: EtOH + silymarin (200 mg / kg body weight) Group 4: EtOH + P3-2:1 (200mg / kg / body weight) Group 5: EtOH + P3-4:1 (200mg / kg body weight) Group 6: EtOH + P3-6:1 (200 mg / kg / body weight).

[0112] The control group and ethanol group were each given 0.9% saline by oral gavage for 28 days.To test the hepatoprotective properties of drug treatment, rats were given silymarin and the synergistic hepatoprotective composition (P3) 30 minutes before ethanol administration for 28 days.

[0113] Composition of animal feed: Protein-17.7%, fat-4.2%, carbohydrates-50.5%, fiber-3.4%, minerals-6.7%, and vitamins-1.7%.

[0114] On day 28 of the study, all animals were anesthetized with chloroform, and blood samples were collected intravenously from control and experimental rats. The blood samples were centrifuged at 3000 rpm for 30 minutes, and serum samples were stored for 20 min until analysis. o The livers of the sacrificed animals were also immediately removed, rinsed appropriately in ice-cold saline, blotted dry, and weighed to record the organ weight. A portion of the liver tissue was then immediately immersed in an appropriate fixative for further histopathological study. The remaining portion of the liver was reserved for biochemical assays.

[0115] Measurement of liver weight relative to body weight At the end of the treatment period, the body weights of all animals were recorded and determined, along with liver weights on the day of sacrifice, which were measured to determine liver weight relative to the final body weight of the animals in each group.

[0116] Preparation of tissue homogenates A 10% w / v liver tissue homogenate was prepared in ice-cold phosphate-buffered saline (PBS) containing 1 mM EDTA (pH 7.4) using a tissue homogenizer. The homogenate was then centrifuged at 10,000 rpm for 30 minutes at 4°C. The supernatant thus obtained was further used for subsequent biochemical assays of tissue oxidative stress markers.

[0117] biochemical analysis Protein content was estimated by the method of Lowry (1951). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (GGT), triglycerides (TG), total cholesterol (TCH), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE) analyses were performed using commercially available diagnostic kits (Micro clinical lab, Tamil Nadu).

[0118] Hepatic ADH activity Alcohol dehydrogenase (ADH) activity was determined by the method of Bonnichsen and Brink (1955). Briefly, ADH activity was measured in 50 mM glycine (pH 9.6), 0.8 mM NAD, 3 mM ethanol, and 50 μL of cytosolic fraction in a final volume of 1 mL. Enzyme activity was measured at 340 nm, and activity was calculated as nmol of NADH formed / min / mg of protein using a molar extinction coefficient of 6.22 × 10 M cm.

[0119] Cytokine evaluation Evaluation of proinflammatory cytokines, namely tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) and interleukin 1 (IL-1), was also performed using commercially available ELISA kits and expressed in picograms per milliliter (pg / mL) (Biovision, Milpitas, CA, USA).

[0120] Histopathological studies Portions of rat liver tissue from control and treated animals were directly fixed in 10% formalin buffer, washed with saline, and dehydrated before being embedded in paraffin. After the entire procedure, 3-5 μm-thick sections were prepared from the tissue blocks using a microtome. The tissue sections were then stained with hematoxylin and eosin (H&E). The tissue sections were observed under a microscope, and corresponding images were captured using a digital camera attached to the microscope.

[0121] statistical analysis Each experiment was repeated at least three times. Data are presented as mean ± SE. The significance of the mean values ​​of various variables between treatment groups was analyzed using a one-way analysis of variance (ANOVA) post hoc test (Tukey's HSD test) after confirming the homogeneity of variance between treatment groups. Statistical tests were performed using SPSS software version 20.0. A value of p<0.05 was considered statistically significant.

[0122] result: A. Effect of the Treated Synergistic Hepatoprotective Composition on Normal Observed Body Weight Table 2 shows the intake of water and food levels in the control and experimental rats. There was no difference in food and water consumption between the control and experimental rat groups during the entire study.

[0123] Tables 3 and 3a show the effects of ethanol and the synergistic hepatoprotective composition (P3) of the present invention on body weight, weight gain, and gain rate, as well as liver weight, in control and experimental rats, respectively. Ethanol-induced rats had significantly decreased body weight and significantly increased liver weight (P<0.05) compared with untreated control (Group 1) rats. Compared with Group 2 rats, liver weights appeared closer to normal in rats (Groups 4-6) treated with the synergistic hepatoprotective composition (P3). Group 2 rats had significantly decreased body weight, weight gain, and gain rate (P<0.05) compared with Group 1 rats. Treatment with the synergistic hepatoprotective composition (P3) of the present invention containing different ratios of curcuminoids and lutein also provided protection against the weight loss induced by ethanol-treated rats, comparable to the effect exhibited by silymarin.

[0124] [Table 2]

[0125] [Table 3]

[0126] B. Effect of the Processed Synergistic Hepatoprotective Composition of the Present Invention on Changes in Liver Function Marker Enzymes Table 4 shows the changes in the activity of liver marker enzymes in control rats and experimental rats. Ethanol administration significantly increased AST, ALT, and GGT levels, respectively (p<0.05). Finally, co-treatment with the inventive processed synergistic hepatoprotective composition (P3) at a dose of 200 mg / kg, containing curcuminoids and lutein in a ratio of 4:1 and 6:1, significantly reduced the elevated levels of AST, ALT, and GGT compared with ethanol-treated rats (Groups 5 and 6) (p<0.05). Furthermore, the results shown by the inventive processed synergistic hepatoprotective composition (P3) containing curcuminoids and lutein in a ratio of 4:1 and 6:1, were comparable to those of silymarin.

[0127] [Table 4]

[0128] Ethanol intake disrupts mitochondrial fatty acid beta-oxidation. In hepatocytes

[0129] [ka]

[0130] Alcohol also increases the ratio of fatty acids to fatty acids in the liver, leading to steatosis. Alcohol also increases lipid transport from the small intestine to the liver, promoting the mobilization of fatty acids from adipose tissue that are taken up by the liver (16). This causes damage to the hepatocyte membrane and leads to elevated blood levels of transaminases (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)). Gamma-glutamyltransferase (GGT) plays an important role in maintaining intracellular homeostasis of oxidative stress, protecting cells from oxidative damage. It is present in the cell membrane and is released into the circulation when the cell membrane is damaged.

[0131] The present findings revealed that ethanol administration caused significant increases in serum AST, ALT, and GGT enzyme activities compared with the control group. These increases may be due to liver damage induced by alcoholism. Administration of the processed synergistic hepatoprotective composition (P3) of the present invention with different ratios of its components (curcuminoids and lutein) effectively alleviated the elevated blood enzyme concentrations caused by alcohol intake, leading to subsequent recovery toward normalization comparable to that of the control group.

[0132] C. Effect of the Processed Synergistic Hepatoprotective Composition of the Present Invention on Ethanol-Induced Changes in Lipid Profile Table 5 shows the comparison of the effect of the processed synergistic hepatoprotective composition (P3) having different ratios of its components (curcuminoids and lutein) on ethanol-induced hepatotoxicity by assessing lipid profile (levels of TG, TCH, LDL-C and HDL-C levels) in control and experimental rats.

[0133] From Table 5, it can be inferred that ethanol treatment also disrupts the homeostasis of lipid profiles in the body, increasing TG, TCH, and LDL-C levels, respectively, while simultaneously decreasing HDL-C levels (p<0.05). Co-treatment with the processed synergistic hepatoprotective composition of the present invention (P3) significantly reversed these changes to near-normal values ​​(p<0.05). During ethanol metabolism, large amounts of reduced nicotinamide adenine dinucleotide (NADH) are produced, thus inhibiting the Krebs cycle and fatty acid oxidation, and supporting hepatic steatosis and serum hyperlipidemia (17).

[0134] Furthermore, the ethanol group also showed an increase in TCH, TG, and LDL levels, with a concomitant decrease in HDL levels. However, treatment with the synergistic hepatoprotective composition (P3) attenuated the above-mentioned changes in lipid profile levels. Therefore, the synergistic hepatoprotective composition (P3) of the present invention is effective in maintaining lipid profile, and is also effective in maintaining healthy cholesterol levels and a healthy LDL / HDL ratio.

[0135] The hypocholesterolemic activity of animals treated with the synergistic hepatoprotective composition (P3) may be attributed to increased hepatic clearance of cholesterol via elevated serum HDL levels or downregulation of cholesterol biosynthetic enzymes (HMG-CoA reductase) ( 18 ).

[0136] [Table 5]

[0137] D. Effect of the Processed Synergistic Hepatoprotective Composition of the Present Invention on Ethanol-Induced Changes in Hepatic ADH, MDA, and 4-HNE Table 6 shows the comparison of the effects of the processed synergistic hepatoprotective composition (P3) of the present invention at different ratios of its components (curcuminoids and lutein at 2:1, 4:1, and 6:1 ratios) on ethanol-induced changes in liver ADH, MDA, and 4-HNE levels. The ethanol-treated group (Group 2) showed a significant increase in ADH, MDA, and 4-HNE levels compared to the control group. The elevated levels of ADH, MDA, and 4-HNE in the ethanol control group indicated the presence of a lipid profile in hepatocytes due to the toxic effects of ethanol.

[0138] Co-treatment with the synergistic hepatoprotective composition of the present invention (P3) containing curcuminoids and lutein in a ratio of 4:1 and 6:1 at a dose of 200 mg / Kg significantly reduced the elevated levels of ADH, MDA and 4-HNE compared to ethanol-treated rats (p<0.05).

[0139] [Table 6]

[0140] E. Effect of the Processed Synergistic Hepatoprotective Composition of the Present Invention on Ethanol-Induced Changes in Cytokine Levels Table 7 shows the effect of the processed synergistic hepatoprotective composition (P3) of the present invention on ethanol-induced changes in cytokine levels. It is clear that administration of ethanol resulted in a significant increase in serum TNF-α, IL-6, and IL-1 levels (p<0.001) compared to control Group 1. Table 7 shows that serum TNF-α, IL-6, and IL-1 levels increased in ethanol-treated Group 2, respectively. Co-treatment with the processed synergistic hepatoprotective composition (P3) caused a significant decrease in serum TNF-α, IL-6, and IL-1 levels, as seen in the results of Groups 4 to 6.

[0141] [Table 7]

[0142] F. Histopathological Analysis of Liver Tissue The histological features shown in Figure 14 demonstrate normal hepatic lobule and cellular structure in the livers of control rats. No pathological changes were observed in the livers of healthy controls. In ethanol-induced rats (i.e., Group 2), the extent of liver injury was assessed by histological analysis after 4 weeks of ethanol intake at 2 gm / kg body weight. For example, dilation of hepatic venous sinuses, extrusion of cords, and atrophy were observed in centrilobular hepatocytes, possibly suggesting hepatocellular regeneration; slight infiltration of mononuclear cells and necrotic cells were observed in the central vein. Vacuolar changes and possibly fatty degeneration were observed in the centrilobular area. Collectively, hepatocyte swelling, edematous degeneration, and vacuolation were prominent in the centrilobular region, reflecting early biochemical and pathological changes resulting from alcoholic liver injury.

[0143] The ethanol-treated rats in Group 2 showed pericentral vein inflammatory reactions accompanied by vacuolar degeneration and necrosis. Groups 3, 4, and 5 showed less vacuolar degeneration and less pericentral vein inflammatory reactions in the liver tissue. The liver tissue in Group 6 showed a nearly normal structure. Thus, the histopathological changes induced by ethanol were significantly improved by the inventive synergistic composition (P3), especially the inventive composition (P3) containing curcuminoids and lutein at a ratio of 4:1 and 6:1 (Groups 5 and 6), which significantly ameliorated hepatic steatosis and showed clear improvement compared to the ethanol-treated groups.

[0144] Test Conclusions In conclusion, in vivo studies demonstrate that the processed synergistic hepatoprotective composition (P3) of the present invention is effective in normalizing and maintaining lipid profiles. It is also effective in maintaining healthy cholesterol levels and a healthy LDL / HDL ratio. Furthermore, the synergistic hepatoprotective composition of the present invention is effective in normalizing and maintaining blood enzyme levels (i.e., AST, ALT, ADH, and GGT). Studies evaluating the lipid peroxidation markers MDA and 4-HNE also demonstrated that the synergistic hepatoprotective composition is effective in reducing lipid peroxidation, which can be caused by various factors, such as unhealthy dietary patterns, high PUFA consumption, and alcohol consumption. Furthermore, the synergistic hepatoprotective composition of the present invention was found to be effective in reducing blood levels of proinflammatory cytokines (TNF-α, IL-6, and IL-1), which are markers of hepatic inflammatory responses. These serum proinflammatory cytokines (TNF-α, IL-6, and IL-1) are thought to be significantly elevated in patients with nonalcoholic and alcoholic fatty liver disease.

[0145] From these results, it is also speculated that the synergistic hepatoprotective composition of the present invention is useful for protecting the liver from ethanol-induced toxicity, possibly through its lipid-lowering and hepatoprotective activities. Furthermore, the composition beneficially improved ethanol-induced hepatotoxicity by maintaining the liver's antioxidant status, reducing the level of lipid profile, the level of liver enzyme markers, and anti-inflammatory activity, as well as for chronic alcohol exposure.

[0146] Histopathological analysis of liver tissues showed that the ethanol-induced changes were significantly improved by the processed synergistic composition of the present invention (P3), especially in the composition of the present invention having curcuminoids and lutein in a ratio of 4:1 and 6:1 (P3).

[0147] While particular embodiments of the synergistic hepatoprotective composition of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made therein without departing from the spirit and scope of the invention, and it is intended that all such changes and modifications be covered in the appended claims.

[0148] References: 1. Akram, M., Shahab-uddin, Ahmed, A., Khan, U., Hannan, A., Mohiuddin, E., Asif, M. (2010) Curcuma Longa and Curcumin: A review Article. Rom. J. Biol. - Plant Biol., Volume 55, No. 2, P. 65-70. 2. Mangels, AR, Holden, JM, Beecher, GR, Forman, MR & Lanza, E. (1993) Carotenoid content of fruits and vegetables: an evaluation of analytic data. J. Am. Diet. Assoc.;93:284-296. 3. Bailey, CA & Chen, BH (1989) Chromatographic analyzes of xanthophylls in egg yolks from laying hens fed turf Bermuda grass (Cynodon dactylon) meal. J. Food Sci.; 54:584-586, 592. 4. Young, AJ & Lowe, GM (2001) Antioxidant and prooxidant properties of carotenoids. Arch. Biochem. Biophys. 385:20-27. 5. Subczynski, W. K., Markowska, E. & Sielewiesiuk, J. (1991) Biochim. Biophys. Acta 1068:68-72. 6. Panasenko, O. M., Sharov, V. S., Briviba, K. & Sies, H. (2000) Interaction of peroxynitrite with carotenoids in human low density lipoproteins. Arch. Biochem. Biophys. 373:302-305. 7. Martin, K. R., Wu, D. & Meydani, M. (2000) The effect of carotenoids on the expression of cell surface adhesion molecules and binding of monocytes to human aortic endothelial cells. Atherosclerosis; 150:265-274. 8. Dwyer, J. H., Navab, M., Dwyer, K. M., Hassan, K., Sun, P., Shircore, A., Hama-Levy, S., Hough, G., Wang, X., Drake, T., Merz, C. N. & Fogelman, A. M. (2001) Oxygenated carotenoid lutein and progression of early atherosclerosis: the Los Angeles Atherosclerosis Study. 9. Iribarren, C., Folsom, A. R., Jacobs, D. R., Gross, M. D., Belcher, J. D. & Eckfeldt, J. H. (1997) Associations of serum vitamin levels, LDL susceptibility to oxidation, and autoantibodies against MDA-LDL with carotid atherosclerosis. Arterioscler. Thromb. Vasc. Biol.; 17:1171-1177. 10. William E. Connor; P. Barton Duell; Ron Kean; Yingming Wang (2007) The Prime Role of HDL to Transport Lutein into the Retina: Evidence from HDL-Deficient WHAM Chicks Having a Mutant ABCA1 Transporter. Investigative Ophthalmology & Visual Science. Vol.48, 4226-4231. 11. Sies H. (1985) Oxidative stress: introductory remarks. In: Sies H, ed. Oxidative Stress. London: Academic Press; 1985:1-8. 12. Pan M, Cederbaum AI, Zhang YL, Ginsberg HN, Williams KJ, Fisher EA. (2004) Lipid peroxidation and oxidant stress regulate hepatic apolipoprotein B degradation and VLDL production. J Clin Invest.; 113: 1277-1287. 13. Esterbauer H, Schaur RJ, Zollner H. (1991) Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med; 11: 81-128. 14. Stewart S, Jones D, Day CP. (2001) Alcoholic liver disease: new insights into mechanisms and preventative strategies.Trends Mol Med; 7(9):408-413. 15. Zhou Z, Wang L, Song Z, Lambert JC, McClain CJ, Kang YJ. (2003) A critical involvement of oxidative stress in acute alcohol-induced hepatic TNF-alpha production.Am J Pathol; 163(3):1137-1146. 16. Shukla I, Azmi L, Gupta SS, Upreti DK, Rao CV. (2018) Melioration of anti-hepatotoxic effect by Lichen rangiferinus against alcohol induced liver damage in rats. J Ayurveda Integr Med. 2018 Jan 29. pii: S0975-9476(17)30047-5. 17. Rukkumani, R., Balasubashini, M.S., Vishwanathan, P. and Menon, V.P. (2002) Comparative effects of curcumin and photo-irradiated curcumin on alcohol-and polyunsaturated fatty acid induced hyperlipidemia. Pharmacol. Res. 46, 257-264. 18. Patil, R.H., Prakash, K. and Maheshwari, V.L. (2011) Hypolipidemic effect of Terminalia arjuna (L.) in experimentally induced hypercholesteremic rats. Acta Biol. Szeged. 55(2), 289-293.

Claims

1. A composition for hepatoprotection comprising curcuminoid and lutein in a ratio ranging from 2:1 to 6:1, The composition is lycopene-free and has a particle size of D ５０ -0.36 μm to 5 μm range, and D ９０ is in the range of 0.60 μm to 10 μm, A composition for liver protection, wherein the curcuminoid and lutein in the composition for liver protection exhibit synergistic activity.

2. 2. The composition of claim 1, wherein the curcuminoid and lutein are present in a ratio ranging from 4:1 to 6:

1.

3. 3. The composition of claim 1 or 2, wherein the curcuminoids and lutein are present in their purified form, the purity of the curcuminoids being in the range of 80-95% and the purity of the lutein being in the range of 70-85%.

4. 4. The composition of any one of claims 1 to 3, wherein the composition is effective in protecting the liver from liver damage or liver toxicity that may be caused by unhealthy eating patterns, high alcohol intake, eating foods with high peroxidized polyunsaturated fatty acid (PUFA) content, or consumption of certain drugs.

5. The composition of claim 4, wherein the composition is effective in protecting against at least one of the following: - Protects the liver from hepatotoxicity by inhibiting oxidative stress damage caused to cells; - Protection against hepatic peroxidation by reducing the production of the thiobarbituric acid reactive species (TBARS) malondialdehyde (MDA) in affected cells; - Protects the liver from hepatotoxicity by increasing the levels of the non-enzymatic antioxidants glutathione reductase (GSH) and superoxide dismutase (SOD) in affected cells; - Protects the liver from hepatotoxicity by preventing reactive oxygen species (ROS) production in affected cells; - Protects the liver from hepatotoxicity by preventing nuclear apoptosis, nuclear fragmentation of cells and depolarization of the mitochondrial membrane of cells in affected cells.

6. 10. The composition of claim 1 or 4, wherein the composition is effective in normalizing and maintaining lipid profiles, maintaining healthy cholesterol levels and a healthy LDL / HDL ratio.

7. 5. The composition of claim 4, wherein the composition is effective in reducing the levels of inflammatory cytokines (TNF-α, IL-6, and IL-1), which is beneficial in reducing the inflammatory response in the liver resulting from alcoholic and non-alcoholic fatty liver disease, which may be caused by unhealthy dietary patterns.

8. The composition of any one of claims 1 to 3, wherein the composition can be taken / administered regularly as a supplement or before, during or after the intake of fatty foods, drugs and / or alcohol.

9. A method for producing a composition for liver protection containing the curcuminoid according to any one of claims 1 to 3 and lutein, comprising: a) mixing curcuminoids and lutein in a predetermined ratio to form a dry blend; b) adding a suitable emulsifier and maltodextrin to the dry blend; c) adding purified water to the dry blend to form a suspension; d) passing the suspension through a liquid colloid mill to form a uniform suspension; e) subjecting the suspension to further processing, including passing it through a homogenizer or high shear particle wet mill, to produce a finely divided emulsion; f) further stirring the micronized emulsion at a speed of 25 rpm for 8 to 12 hours to bring the mass temperature to a temperature of 25°C to 40°C; g) subjecting the micronized emulsion to further processing, including concentration and / or drying, to obtain a hepatoprotective composition; The resulting hepatoprotective composition may be further passed through an appropriate particle sieve to obtain a uniform final product; The hepatoprotective composition contains curcuminoid and lutein in a ratio ranging from 2:1 to 6:1, and the particle size of the composition is D ５０ -0.36 μm to 5 μm range, and D ９０ is in the range of 0.60 μm to 10 μm.

10. 10. The method of claim 9, wherein curcuminoids can be obtained / extracted and purified from the plant Curcuma longa and lutein can be obtained / extracted and purified from the marigold plant Tagetes erecta or any other suitable plant source.

11. 4. The composition of any one of claims 1 to 3, wherein the composition is formulated and used in various food and beverage products, teas, infusions, fruit juices, beverages, milk and milk products, cereal-based products, alcoholic beverages and processed foods, as a dietary supplement, pharmaceutical, dietary / nutraceutical or therapeutic / health ingredient.

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