TWI810644B - Therapeutic herbal composition - Google Patents

Abstract

The present invention relates to a therapeutic herbal composition comprising of a novel combination of the phytochemicals namely curcuminoids and carotenoids such as lutein. More particularly the therapeutic herbal composition comprises of Curcuminoids and Lutein along with the excipients wherein the curcuminoids are present in the range of 24- 49%, lutein is present in the range of 1%-14.5% and the excipients are in the range of 50-64%. The said therapeutic herbal composition is effective in regulating blood lipid profile, in modulating the inflammatory effects and having anti-inflammatory effects. Additionally, the said therapeutic herbal composition is effective in management of diseases caused due to factors such as inflammation, oxidative stress etc. and diseases such as Diabetic Retinopathy and Age Related Macular Degeneration. Also provided is a novel process for manufacturing such a composition and its use in foods, beverages including alcoholic beverages and as nutraceutical products, dietary supplements and pharmaceuticals.

Description

therapeutic herbal composition

The present invention relates to a therapeutic herbal composition comprising a novel combination of botanical compounds, namely curcuminoids and carotenoids such as lutein. More particularly, the present invention relates to a therapeutic herbal composition comprising curcuminoids and lutein. The therapeutic herbal compositions of the present invention are beneficial to the general health of humans. The therapeutic herbal composition effectively regulates blood lipid profile and modulates inflammatory effects. The invention also provides a method for the manufacture of the composition and the use of the composition in food, beverages (alcoholic beverages) and as nutraceuticals, dietary supplements and medicines.

Inflammation is the immune system's response to protect an organism from infection and injury and works by removing damaged tissue components so the body can begin healing. Inflammation that lasts only a few days is called acute inflammation, and one that lasts longer is called chronic inflammation. Infectious agents/pathogens such as viruses and bacteria are some of the most common inflammatory stimuli. Other factors that can stimulate inflammation include physical agents, chemicals, inappropriate immune responses, and tissue death. Acute and chronic inflammation-mediated tissue damage has been observed in many organ systems, including heart, pancreas, liver, kidney, lung, brain, gut, and reproductive system. Inflammation is a common pathogenesis of many chronic diseases, including cancer, atherosclerosis, insulin resistance, type 2 diabetes, and cardiovascular disease.

Although the course of the inflammatory response depends on the precise nature of the initial stimulus and its However, they all share a common mechanism, which can be summarized as follows: 1) recognition of noxious stimuli by cell surface pattern receptors; 2) activation of inflammatory pathways; 3) release of inflammatory markers; and 4) recruitment of inflammatory cells. Inflammatory pathways influence the pathogenesis of many chronic diseases and involve common inflammatory mediators and regulatory pathways. Various chemical mediators are released from the circulatory system, inflammatory cells and damaged tissues to regulate the inflammatory response. Chemical mediators released include (1) vasoactive amines such as histamine and serotonin, (2) peptides (e.g., bradykinin), and (3) eicosanoids (e.g., thromboxanes, leukotrienes, and prostaglandins ). Prostaglandin E2 (PGE2), a member of the eicosanoid family of regulatory molecules, is produced in large quantities at inflamed sites and is involved in all processes leading to the typical signs of inflammation: redness; swelling and pain. Redness and edema result from increased blood flow into inflamed tissue.

Various studies have shown that inflammatory cytokines play an important role in various inflammatory diseases. Interleukins (such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)) through the interaction with TLR, IL-1 receptor (IL- 1R), IL-6 receptor (IL-6R), and TNF receptor (TNFR) interact to mediate inflammation. Receptor activation triggers important intracellular signaling pathways including mitogen-activated protein kinase (MAPK), nuclear factor kappa-B (NF-κB), and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) way. The tumor necrosis factor-α (TNF-α) is a cytokine produced by various cell types including macrophages, neutrophils and lymphocytes.

Nitric oxide (NO) is a signaling molecule that plays a key role in the pathogenesis of inflammation. Nitric oxide produces anti-inflammatory effects under normal physiological conditions. Nitric oxide, on the other hand, induces inflammation due to its overproduction in abnormal conditions. Nitric oxide has been associated with acute and chronic inflammatory disorders, including various types of arthritis, gastrointestinal disorders, inflammatory disorders of the central nervous system, and certain forms of asthma, and is considered a pro-inflammatory marker. In vivo production of NO is mediated by the nitric oxide synthase (NOS) enzyme family, including inducible nitric oxide synthase (I-NOS), which is mediated by many different immune stimulants, including lipopolysaccharide (LPS), Interferon gamma and interleukin-1 (IL-1)) activation. Therefore, NO inhibitors are considered useful in the treatment and/or control of inflammatory disorders. Natural products provide a large number of NO inhibitors. The disease preventive capabilities of fruits and vegetables have been attributed to the antioxidants/polyphenols present in these dietary sources.

Inflammation of the liver protects this organ from infection and injury, but excessive inflammation can lead to massive loss of hepatocytes, ischemia-reperfusion injury, metabolic changes and eventually permanent liver damage. Inflammation can destroy liver parenchymal cells, increasing the risk of chronic liver diseases such as non-alcoholic fatty liver disease (NAFLD) or viral hepatitis. The liver is a vital organ and plays an important role in the metabolism and detoxification of various endogenous and exogenous harmful substances. Structural changes of the drug occur in the liver leading to bioactive or inactive metabolites and some of these are toxic. Thus, the liver is a vulnerable target for damage from various chemicals and drugs. The main causes of liver disease are toxic chemicals, excessive alcohol consumption, infections and autoimmune diseases.

In addition, various inflammatory stimuli produced during oxidative metabolism (such as excess reactive oxygen species (ROS)/reactive nitrogen species (RNS)) and some natural or artificial chemicals have been reported to initiate the synthesis and secretion of pro-inflammatory cytokines the inflammatory process. Oxidative stress has been implicated in the pathogenesis of various diseases such as cardiovascular disease, cancer, diabetes, hypertension, aging and atherosclerosis. Oxidative stress refers to the imbalance between the production of ROS and oxidants and the counteracting activity of antioxidants. Oxidative stress has been considered a major factor in the initiation and progression of liver injury. Various risk factors such as alcohol and drugs are known to induce oxidative stress which in turn can lead to chronic diseases such as fatty liver disease. Fatty liver disease includes alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD). Accumulating evidence indicates that oxidative stress caused by generation of reactive oxygen species (ROS) plays a key role in the progression of ALD and NAFLD. Excessive production of ROS has deleterious effects in cellular functions, ultimately leading to fatty liver disease. In mitochondria, increased ROS production can cause mtDNA depletion, attack biomolecules (ie, proteins, carbohydrates, and lipids) and damage mitochondrial membrane. Notably, mitochondria have substantial concentrations of phospholipids containing polyunsaturated fatty acids (PUFA). These PUFAs are more susceptible to oxidative damage due to the presence of double bonds in their chemical structures, which lead to lipid peroxidation. PUFA peroxidation enhances post-endoplasmic reticulum post-presecretory proteolysis of ApoB, thereby reducing very low-density lipoprotein (VLDL) secretion; this can further promote triglyceride (TG) accumulation in the liver. Furthermore, aldehydes formed by PUFA peroxidation impair cellular homeostasis, as these molecules affect nucleotide and protein synthesis, reduce hepatic glutathione content and increase production of the pro-inflammatory interleukin TNF-α. These effects lead to liver cell death and necrosis, inflammation and liver fibrosis.

Liver injury occurs through several interrelated pathways. Alcohol metabolism occurs primarily in the liver. The main pathway of ethanol metabolism is the dehydrogenase system. Alcohol dehydrogenase and acetaldehyde dehydrogenase reduce nicotinamide adenine dinucleotide (NAD) to NADH (the reduced form of NAD). Changes in the NAD/NADH ratio promote fatty liver by inhibiting gluconeogenesis and fatty acid oxidation. Chronic alcohol exposure also activates hepatic macrophages, which then produce tumor necrosis factor-α (TNF-α). TNF-α induces mitochondria to increase the production of reactive oxygen species (ROS). This oxidative stress promotes hepatocyte necrosis and apoptosis.

Therefore, oxidative stress products can also be used as markers of inflammatory responses.

Diabetic retinopathy (DR) is a complication of diabetes that affects the eyes, causing visual disability and blindness. Often accompanied by lipid exudation. Dyslipidemia leads to hard exudates and the development of clinically significant macular edema (CSME). These further interfere with vision. Elevated lipid concentrations are associated with endothelial dysfunction, which appears to play an important role in the pathogenesis of diabetic retinopathy, particularly associated with disruption of the blood-retinal barrier. Recently, analysis of lipid profiles in Diabetes Control and Complications Trial (DCCT) samples established a strong association between the development of DR and dyslipidemia in type 1 diabetes, and several clinical trials using lipid-lowering drugs showed an association between dyslipidemia and the progression of DR in type 2 diabetes (Action to Control Cardiovascular Risk in Diabetes Follow-On, 2016, Frank, 2014, Matthews, 2011, Wright and Dodson, 2011).

The lutein carotenoids lutein and zeaxanthin are found in and around the macula of the primate retina, where they are known as the macular pigment (MP). Dietary lutein and zeaxanthin are absorbed with fat in the gut and are eventually transported to the liver. Intrahepatic lutein and zeaxanthin are incorporated into lipoprotein molecules, which are transported throughout the bloodstream as part of carotenoid-lipoprotein complexes. Both MP and serum lipoproteins have been associated with the risk of neurodegenerative diseases such as age-related macular degeneration (AMD). AMD is a chronic, progressive, degenerative eye disease that affects the central retina responsible for highest vision. It has become the leading cause of legal blindness among older adults in both developed and developing countries. Both high-density lipoprotein (HDL) (associated with reduced risk of AMD) and low-density lipoprotein (LDL) (associated with increased risk) carry lutein and zeaxanthin. Therefore, it has been reported that serum lutein, retinal lutein, and lipoprotein concentrations are significantly correlated, and changes in lipoprotein concentrations can affect retinal lutein concentrations. Oxidative stress associated with low-grade inflammation and hypoxia in the outer retina has also been shown to be important in the pathogenesis of AMD. This is consistent with the idea that low macular pigment optical density (MPOD) may be a risk factor for AMD, since macular carotenoids exhibit potent properties as antioxidants.

Therefore, limiting the inflammatory process by using anti-inflammatory agents to inhibit and/or modulate one or more of the inflammatory mediators described above is important to control this process and limit the diseases caused by it. However, widely used synthetic anti-inflammatory agents/drugs appear to have side effects with long-term use. Therefore, there remains a need for newer and better anti-inflammatory agents from natural products.

In addition, as described above, maintaining the lipid profile of the body is also important to prevent liver damage (the liver is a vital organ) and thereby help control Diseases caused by hormones, and diseases such as diabetic retinopathy and age-related macular degeneration.

Phytochemicals are biologically active compounds found in plants that are used in food and medicine. Phytochemicals such as curcuminoids and carotenoids such as lutein and zeaxanthin are known for their health-promoting properties.

Turmeric ( Curcuma longa ) is a popular Indian spice, which is a perennial rhizome herbaceous plant belonging to the ginger family ( Zingiberaceae ), which is native to the south subtropical region. Turmeric is primarily known for its use in Indian cuisine as a common ingredient in curries and other ethnic cuisines. Turmeric has also been used for centuries in Ayurvedic medicine, which combines the medicinal properties of the herb with food. In the Ayurvedic system of medicine, turmeric has been assigned many therapeutic activities for use in a wide range of diseases and conditions, including those of the skin, lungs, gastrointestinal system, aches, pains, wounds, sprains and liver diseases. Curcuminoids are polyphenolic compounds in turmeric rhizomes and are responsible for the yellow color of turmeric. Curcuminoids make up 2 to 4% of dry turmeric root powder. Curcumin is the main curcuminoid of turmeric, and the other two curcuminoids are demethoxycurcumin and bisdemethoxycurcumin. The biological effects of curcumin range from anti-oxidative, anti-inflammatory to anti-angiogenesis and have also been shown to have specific anti-tumor activity. Turmeric has been found to have antioxidant properties, as well as its ability to reduce the formation of pro-inflammatory cytokines.

Curcumin may be administered alone in the form of turmeric, turmeric concentrate, curcuminoid powder, substantially pure (95%) curcuminoid, or curcuminoids. A problem that greatly limits the effectiveness and utility of curcumin is its low bioavailability due to water insolubility, instability at physiological pH, photodegradation, slow cellular uptake and rapid metabolism to inactive metabolites. The water solubility of curcumin is estimated to be 11 ng/mL (https://pubchem.ncbi.nlm.nih.gov/compound/curcumin).

Pharmacokinetic studies in animals have demonstrated that 40 to 85% of oral doses of turmeric Flavonoids pass through the gastrointestinal tract unchanged, and most absorbed flavonoids are metabolized in the intestinal mucosa and liver. Numerous pharmacokinetic studies in humans and rodents have shown that curcumin has very low bioavailability, limiting its benefits. This low bioavailability is likely due to its low absorption and rapid metabolism and rapid elimination in the gut and liver. Due to curcumin's low absorption rate, curcumin is often formulated with other plant compounds to increase absorption and enhance anti-inflammatory effects.

Phase I clinical trials have shown that curcumin is safe in humans even at high doses (12g/day), but shows poor bioavailability. Potential factors that limit the bioavailability of curcumin include poor absorption, rapid metabolism, and rapid systemic elimination. The bioavailability of curcumin ingested in food can be increased due to cooking or dissolving in oil. A number of approaches have been initiated to improve bioavailability. These methods involve firstly using an adjuvant (such as piperine) that interferes with glucuronidation; secondly, using liposomal curcumin; thirdly, using curcumin nanoparticles; fourthly, using curcumin phospholipid complexes; and fifthly, using Structural analogs of curcumin (eg, EF-24) are used. The latter is reported to be rapidly absorbed, with a peak plasma half-life. Despite their low bioavailability, curcuminoids have documented therapeutic utility against various human diseases including cancer, cardiovascular disease, diabetes, arthritis, neurological disorders and Crohn's disease. Nanoparticles, liposomes, micelles, and phospholipid complexes are other promising novel formulations that appear to offer longer circulation, better permeability, and resistance to metabolic processes. There is therefore an ongoing need to enhance the bioavailability of curcumin to gain greater benefit from the therapeutic properties of curcumin for the treatment of human diseases.

Lutein belongs to a large class of plant pigments, usually its isomer zeaxanthin and trace amounts of other carotene (such as β-carotene) and cryptoxanthin, collectively referred to as carotenoids. Its presence in human tissues is solely due to ingestion of plant sources; it is not synthesized by animal tissues. Lutein is found in a wide range of fruits and vegetables and imparts its yellow color to plants such as corn. Lutein is found in particularly high concentrations in green leafy vegetables such as spinach, kale and kale. Lutein is also present in some animal products, such as egg yolks, due to the consumption of plant products by animals. Lutein and zeaxanthin are found together in many food sources. Dark green leafy vegetables are the main source of lutein and zeaxanthin, but they are also present in smaller amounts in other colored fruits and vegetables (such as broccoli, orange peppers, corn, peas, persimmons, and tangerines) . Lutein is usually isolated from marigold oleoresin together with zeaxanthin. Of the 20 to 30 carotenoids found in human blood and tissues, only lutein and zeaxanthin are found in the lens and retina. Lutein and zeaxanthin are concentrated in the macula, or central area of the retina, and are called macular pigments.

Various studies have shown that lutein can reduce the risk of developing two of the most common eye diseases (ie, cataracts and macular degeneration) in older adults. Oxidative stress in the eye is high due to intense light exposure and a high rate of oxidative metabolism in the retina. The antioxidant properties of lutein may reduce the extent to which oxidative damage contributes to these diseases or may minimize damage due to oxidative stress by limiting the extent to which oxygen permeates membranes. Furthermore, an association between the highest serum or dietary lutein concentrations and a lower incidence of coronary heart disease or stroke has been found between individuals. In two epidemiological studies, individuals with the highest serum concentrations of lutein plus zeaxanthin had a significantly lower risk of developing coronary heart disease, as measured by carotid intima thickness. Lutein and zeaxanthin are mainly transported in plasma by high-density lipoprotein (HDL).

Thus, as discussed above, curcumin and lutein have been shown to be antioxidants and contribute to diseases due to oxidative stress. Therefore, it would be advantageous to prepare compositions comprising curcuminoids from curcumin and carotenoids, such as lutein and zeaxanthin, which would have potentially beneficial effects in modulating lipid profiles by inhibiting and /or regulate the production of one or more key inflammatory mediators to have an anti-inflammatory effect.

Therefore, the purpose of the present invention is to provide an effective regulation of blood lipid profile and has Therapeutic composition for anti-inflammatory effect. In addition, the therapeutic herbal composition is effective in controlling diseases due to factors such as inflammation (including inflammation due to pathogenic infection), oxidative stress, and diseases such as diabetic retinopathy and age-related macular degeneration.

It is also an object of the present invention to provide a therapeutic herbal composition comprising curcuminoids and carotenoids such as lutein and zeaxanthin which shows a synergistic effect of its ingredients at very low doses and At the same time, it has high bioavailability.

The present invention relates to a therapeutic herbal composition comprising a novel combination of botanical compounds, namely curcuminoids and carotenoids such as lutein. More particularly, the therapeutic herbal composition comprises curcuminoids and lutein. The therapeutic herbal composition is effective in promoting health, more particularly in regulating blood lipid profile and has anti-inflammatory effect. The anti-inflammatory effect is produced by inhibiting and/or modulating the production of one or more key inflammatory mediators. In addition, the therapeutic herbal composition effectively protects the liver from any oxidative damage, including damage caused by peroxidized polyunsaturated fatty acids, thereby helping to control diseases due to factors such as inflammation, oxidative stress, and diseases such as diabetes Retinopathy and age-related macular degeneration. The invention also provides a novel process for the manufacture of the composition and the use of the composition in food, beverages (alcoholic beverages) and as nutraceuticals, dietary supplements and pharmaceuticals.

According to one embodiment of the present invention, there is provided a therapeutic herbal composition comprising the botanical compounds curcuminoids and lutein.

According to one embodiment of the present invention, curcuminoids may be used in the form of curcuminoid powder or curcuminoid concentrate and lutein may be used in the form of lutein crystals or lutein concentrate. Furthermore, the term "lutein" as used herein includes mixtures of lutein and zeaxanthin, since zeaxanthin is usually present/isolated together with lutein.

According to an embodiment of the present invention, the therapeutic herbal composition comprises curcumin powder/concentrate in the range of 24 to 49%, lutein crystals/concentrate in the range of 1 to 14.5% and Excipients in the range of 50 to 64%. Furthermore, the curcuminoid powder/concentrate may have a curcuminoid content of 80 to 95% and the lutein crystals/concentrate may have a lutein content of 70 to 80% and a zeaxanthin content of 4.5 to 9% .

According to one embodiment of the present invention, curcuminoids and lutein are present in the therapeutic herbal composition in their purified form.

According to one embodiment of the present invention, the curcuminoid powder/concentrate can be obtained/extracted and purified from the plant Curcuma longa and the lutein crystals/concentrate can be obtained from the marigold plant Calendula ( Tagetes erecta ) or any other suitable plant source Obtained/extracted and purified.

According to another embodiment of the present invention, the therapeutic herbal composition is effective in down-regulating inflammatory pathways. The composition also reduces the formation of reactive oxygen species (ROS) and balances the NADH/NAD (nicotinamide adenine dinucleotide (NAD) to NADH (reduced form of NAD)) ratio. In addition, the therapeutic herbal composition of the present invention is also effective in controlling fatty liver formation.

According to another embodiment of the present invention, the therapeutic herbal composition is effective in regulating blood lipid profile and has anti-inflammatory effect. Some contributing factors to abnormal lipid profiles and inflammation in the body can be unhealthy eating patterns, high alcohol intake, intake of foods with high peroxidized polyunsaturated fatty acid (PUFA) content, or due to the use of certain medications. Inflammation in the body can also be caused by pathogenic infections caused by infectious agents/pathogens such as viruses (including viruses such as coronavirus disease (COVID-19) and other infection-causing viruses) and bacteria. The anti-inflammatory effect is produced by inhibiting and/or modulating the production of one or more key inflammatory mediators. In addition, the therapeutic herbal composition of the present invention is also effective in protecting the liver from non-alcoholic fatty liver disease caused by unhealthy dietary patterns.

According to another embodiment of the present invention, the therapeutic herbal composition is effective in inhibiting oxidative stress damage in infected cells.

According to another embodiment of the present invention, the therapeutic herbal composition helps to protect the liver from hepatic peroxidation. The same conclusion has been established through the effect studies conducted by the inventors in vitro and in vivo.

According to yet other embodiments of the present invention, the therapeutic herbal composition is effective in modulating blood lipid profile. In addition, the therapeutic herbal composition is effective in maintaining healthy cholesterol concentrations and healthy low-density lipoprotein to high-density lipoprotein (LDL/HDL) ratios. The composition is also effective in lowering elevated serum triglycerides. Since the therapeutic herbal composition is effective in modulating the blood lipid profile, it is also effective in controlling diseases such as diabetic retinopathy and age-related macular degeneration because the therapeutic herbal composition results in a rapid increase in macular pigment optical density (MPOD).

According to another embodiment of the present invention, the therapeutic herbal composition is effective to increase the concentration of non-enzymatic antioxidants glutathione reductase (GSH) and superoxide dismutase (SOD) in infected cells.

According to another embodiment of the present invention, the therapeutic herbal composition is effective in reducing the production of thiobarbituric acid reactive substances (TBARS) malondialdehyde (MDA) in infected cells as shown in in vitro studies, wherein the Therapeutic herbal composition is effective against hepatic peroxidation.

In one embodiment of the present invention, the therapeutic herbal composition of the present invention is effective in preventing nuclear apoptosis and nuclear fragmentation in infected cells, as in in vitro studies.

In one embodiment of the present invention, the therapeutic herbal composition of the present invention effectively reduces the concentration of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1), which is beneficial to reduce the A large amount of alcohol, intake of food containing a large amount of peroxidized polyunsaturated fatty acids (PUFA) or due to taking certain drugs, due to pathogenic infection, and due to alcoholic And liver inflammation caused by non-alcoholic fatty liver disease.

In one embodiment of the present invention, the therapeutic herbal composition of the present invention exhibits lipid-lowering activity, thus it is also effective against liver toxicity that may be caused by alcohol consumption, drugs and unhealthy dietary patterns.

According to another embodiment of the present invention, the therapeutic herbal composition may be taken/ingested as a supplement on a regular basis or before, during or after intake of fatty foods, drugs and/or alcohol.

According to another embodiment of the present invention, there is provided a method for the manufacture of a therapeutic herbal composition comprising the botanical compounds curcuminoids and lutein, wherein the method comprises: a) using a curcuminoid content of 80 to 95% Curcuminoid powder/concentrate in the range and lutein crystals/concentrate with lutein content in the range of 70 to 80% and zeaxanthin content in the range of 4.5 to 9% and mix the curcuminoid powder in a predetermined amount /concentrate and lutein crystals/concentrate to form a blend; the particle size of the untreated dry blend may fall within the range of D ₅₀ -20 μm to 50 μm and D ₉₀ -100 μm to 200 μm; b) excipient Add agents such as suitable emulsifiers and maltodextrin to the blend; c) add purified water to the blend to make a suspension; d) pass the suspension through a liquid colloid mill to form a homogeneous suspension e) subjecting the suspension to further processing such as through a homogenizer or a high shear particle wet mill to produce a micronized emulsion; f) further agitating the micronized emulsion at a speed of 25 rpm for 8 to 12 hours to raise the temperature of the mass to a temperature of 25°C to 40°C, preferably 25°C to 30°C; g) subjecting the micronized emulsion to further processing such as concentration and/or drying to obtain the therapeutic herbal composition.

The method further comprises passing the dried therapeutic herbal composition through a suitable particle sieve to obtain a homogeneous finished product. The particle size of the treated therapeutic herbal composition may fall within the range of D ₅₀ -0.36 μm to 5 μm and D ₉₀ in the range of 0.60 μm to 10 μm. This particle size reduction facilitates high bioavailability of the therapeutic herbal composition.

According to one embodiment of the present invention, the predetermined amount of curcuminoid powder/concentrate and lutein crystals/concentrate can be such that the curcuminoid powder/concentrate is in the range of 24 to 49%, lutein crystals /Concentrate in the range of 1 to 14.5% and excipient in the range of 50 to 64%.

According to still other embodiments of the present invention, the final processed therapeutic herbal composition comprises curcuminoids in the range of 22.5% to 46.5%, lutein in the range of 0.90% to 11.25%, and lutein in the range of 0.06% to 1.12% zeaxanthin. It is apparent that the amount of zeaxanthin in the final composition constitutes approximately 6 to 9% of the total content of lutein in the final composition.

According to one embodiment of the present invention, the excipients used are maltodextrin and emulsifier.

According to another embodiment of the present invention, the therapeutic herbal composition can be added to food and/or beverage products or formulated as a dietary supplement.

According to another embodiment of the present invention, the therapeutic herbal 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 therapeutic herbal composition can be used as a health ingredient include, but are not limited to, teas, infusions, fruit juices, beverages, milk and milk products, cereal products, alcoholic beverages, processed foods, and the like.

According to another embodiment of the present invention, the therapeutic herbal composition can also be formulated into a suitable dosage form selected from the group consisting of powder, paste, lozenge, syrup and/or capsule, etc.

According to another embodiment of the present invention, the botanical compounds curcuminoids and lutein exhibit synergistic activity in the therapeutic herbal composition. Furthermore, the curcuminoids and luteinoids present in the therapeutic herbal composition are highly bioavailable when compared to the bioavailability of higher doses of unformulated lutein and unformulated curcuminoids. Furthermore, these curcuminoids and lutein are present in amounts well below the recommended daily requirement.

The above and other features and aspects of the invention are more clearly described in the full specification.

The present invention can be described according to the following figure, wherein: Figure 1(a) illustrates the cytotoxic effect of different concentrations of untreated curcuminoid (U1) in human liver cancer (HepG2) cells.

Figure 1(b) illustrates the cytotoxic effect of different concentrations of untreated lutein (U2) in human liver cancer (HepG2) cells.

Figure 1(c) illustrates the cytotoxic effect of different concentrations of untreated therapeutic herbal composition (U3) in human liver cancer (HepG2) cells.

Figure 1(d) illustrates the cytotoxic effect of different concentrations of processed curcuminoids (P1) in human liver cancer (HepG2) cells.

Figure 1(e) illustrates the cytotoxic effect of different concentrations of processed lutein (P2) in HepG2 cells.

Figure 1(f) illustrates the cytotoxic effect of different concentrations of the treated therapeutic herbal composition (P3) in HepG2 cells.

Figure 2(a) illustrates the cytotoxic effect of different concentrations of untreated curcuminoid (U1) in ethanol-induced HepG2 cells.

Figure 2(b) illustrates the cytotoxic effect of different concentrations of untreated lutein (U2) in ethanol-induced HepG2 cells.

Figure 2(c) illustrates the cytotoxic effect of different concentrations of untreated therapeutic herbal composition (U3) in ethanol-induced HepG2 cells.

Figure 2(d) illustrates the cytotoxic effect of different concentrations of processed curcuminoids (P1) in ethanol-induced HepG2 cells.

Figure 2(e) illustrates the cytotoxic effect of different concentrations of processed lutein (P2) in ethanol-induced HepG2 cells.

Figure 2(f) illustrates the cytotoxic effect of different concentrations of the treated therapeutic herbal composition (P3) in ethanol-induced HepG2 cells.

Figure 3(a) illustrates the therapeutic effect of untreated and treated therapeutic herbal compositions on the activity of thiobarbituric acid-responsive substances (TBARS) in ethanol-induced human liver cancer (HepG2) cells.

Figure 3(b) illustrates the therapeutic effect of untreated and treated therapeutic herbal compositions on superoxide dismutase (SOD) activity in ethanol-induced human liver cancer (HepG2) cells.

Figure 3(c) illustrates the therapeutic effect of untreated and treated therapeutic herbal compositions on the concentration of glutathione reductase (GSH) in ethanol-induced HepG2 cells.

Figure 4(a) is a bar graph showing the cytotoxic effect of treated therapeutic herbal compositions in acetaminophen (APAP)-treated HepG2 cells.

Figure 4(b) is a bar graph showing the cytotoxic effect of silymarin in APAP-treated HepG2 cells.

Figure 5(a) illustrates a comparison of the therapeutic effects of treated therapeutic herbal compositions compared to silymarin on TBARS activity in APAP-treated HepG2 cells.

Figure 5(b) illustrates a comparison of the therapeutic effects of treated therapeutic herbal compositions compared to silymarin on SOD activity in APAP-treated HepG2 cells.

Figure 5(c) illustrates a comparison of the therapeutic effects of treated therapeutic herbal compositions compared to silymarin on GSH concentration in APAP-treated HepG2 cells.

Figure 6 illustrates a comparison of the effect of untreated and treated therapeutic herbal compositions compared to silymarin on ethanol-induced intracellular reactive oxygen species (ROS) production in HepG2 cells.

Figure 7 illustrates a comparison of the effect of untreated and treated therapeutic herbal compositions compared to silymarin on ethanol-mediated reduction of mitochondrial membrane potential (MMP) in HepG2 cells.

Figure 8 illustrates a comparison of the effect of untreated and treated therapeutic herbal compositions compared to silymarin on ethanol-induced nuclear apoptosis in HepG2 cells.

Figure 9 illustrates a comparison of the effect of untreated and treated therapeutic herbal compositions compared to silymarin on ethanol-induced nuclear fragmentation in HepG2 cells.

Figure 10 illustrates a comparison of the effect of treated therapeutic herbal compositions compared to silymarin on APAP-induced intracellular ROS production in HepG2 cells.

Figure 11 illustrates a comparison of the effect of treated therapeutic herbal compositions compared to silymarin on APAP-mediated MMP reduction in HepG2 cells.

Figure 12 illustrates a comparison of the effect of treated therapeutic herbal compositions compared to silymarin on APAP-induced nuclear apoptosis in HepG2 cells.

Figure 13 illustrates a comparison of the effect of treated therapeutic herbal compositions compared to silymarin on APAP-induced nuclear fragmentation in HepG2 cells.

Figure 14 illustrates the effect of treated therapeutic herbal composition compared to silymarin on alcohol-induced histopathological changes in rat liver at day 28 of the study.

Figure 15 shows mean plasma concentration-time profiles of curcuminoids.

Figure 16 shows the mean plasma concentration-time profile of lutein.

Some representative embodiments of the invention are discussed below. The invention in its broader aspects is not limited to the specific details and representative methods. Illustrative examples are described in this section in conjunction with the embodiments and methods provided herein. The invention in its various aspects is particularly pointed out and distinctly claimed in the appended claims to be read in light of this specification.

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

All amounts expressed in "%" mean percent by weight of the total solution or composition unless otherwise specified.

The term "lutein" as used herein includes mixtures of lutein and zeaxanthin, since zeaxanthin is usually present/isolated together with lutein.

All references cited are hereby incorporated by reference in their entirety. Citation of any reference is not an admission with any certainty as to its applicability as prior art to the present invention.

Unless clearly defined otherwise, technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which this invention belongs. Reference is made herein to various methodologies and materials known to those skilled in the art.

Any suitable materials and/or methods known to those skilled in the art may be used to further carry out the invention. However, preferred materials and methods are described herein. Materials, reagents and the like to which reference is made in the following specification and examples are available from commercial sources unless otherwise mentioned.

The present invention is described in detail as follows in its product and method aspects.

The present invention relates to a therapeutic composition comprising a novel combination of botanical compounds, namely curcuminoids and carotenoids such as lutein. More particularly, the therapeutic herbal composition comprises curcuminoids and lutein. The therapeutic herbal composition effectively regulates blood lipid profile and modulates inflammatory effects. Such modulation of inflammatory or anti-inflammatory effects occurs by inhibiting and/or modulating the production of one or more key inflammatory mediators. In addition, the therapeutic herbal composition effectively protects the liver from any oxidative damage, including damage caused by peroxidized polyunsaturated fatty acids, thereby helping to control diseases due to factors such as inflammation, oxidative stress, and diseases such as diabetes Retinopathy and age-related macular degeneration. The invention also provides a novel process for the manufacture of the composition and the use of the composition in food, beverages (including alcoholic beverages) and nutritional products and/or as a dietary supplement.

According to one embodiment of the present invention, the therapeutic herbal composition comprises curcuminoids and lutein.

According to one embodiment of the present invention, curcuminoids may be used in the form of curcuminoid powder or curcuminoid concentrate and lutein may be used in the form of lutein crystals or lutein concentrate.

According to an embodiment of the present invention, the therapeutic herbal composition comprises curcumin powder/concentrate in the range of 24 to 49%, lutein crystals/concentrate in the range of 1 to 14.5%, and Excipients in the range of 50 to 64%. Furthermore, the curcuminoids may have a curcuminoid content of 80 to 95% and the lutein crystals/concentrate may have a lutein content of 70 to 80% and a zeaxanthin content of 4.5 to 9%.

According to one embodiment of the present invention, curcuminoids and lutein are present in the therapeutic herbal composition in their purified form.

According to one embodiment of the present invention, the curcuminoid powder/concentrate can be obtained/extracted and purified from the plant Curcuma longa and the lutein crystals/concentrate can be obtained/extracted from the marigold plant Calendula officinalis or any other suitable plant source and purification.

According to another embodiment of the present invention, the therapeutic herbal composition is effective in down-regulating inflammatory pathways and is also effective in modulating blood lipid profile. Some contributing factors to abnormal lipid profiles and inflammation in the body may be unhealthy eating patterns, high alcohol consumption, certain medications, or foods high in peroxidized polyunsaturated fatty acids (PUFAs) in the diet. Inflammation in the body can also be caused by pathogenic infections caused by infectious agents/pathogens such as viruses (including viruses such as coronavirus disease (COVID-19) and other infection-causing viruses) and bacteria. The anti-inflammatory effect is produced by inhibiting and/or modulating the production of one or more key inflammatory mediators. In addition, the therapeutic herbal composition of the present invention is also effective in protecting the liver from non-alcoholic fatty liver disease caused by unhealthy dietary patterns.

According to another embodiment of the present invention, the therapeutic herbal composition has an inhibitory effect on oxidative stress damage in cells. The inventors have studied that the therapeutic herbal composition is effective against oxidative stress damage and hepatic peroxidation in liver cells, thereby indicating that the therapeutic herbal composition inhibits oxidative stress damage in infected cells, which can be caused by various factors Such as alcohol intake, drug intake, or unhealthy dietary patterns (including high intake of peroxidized polyunsaturated fatty acids PUFA in the diet, etc.).

According to another embodiment of the present invention, the therapeutic herbal composition is effective in reducing the production of thiobarbituric acid reactive substances (TBARS) malondialdehyde (MDA) in infected cells as shown in in vitro studies, wherein it was found that The therapeutic herbal composition is effective against hepatic peroxidation.

According to another embodiment of the present invention, the therapeutic herbal composition is effective to increase the concentration of non-enzymatic antioxidants glutathione reductase (GSH) and superoxide dismutase (SOD) in infected cells. The reduction of these non-enzyme antioxidants can be caused by various factors such as alcohol intake, drug intake or unhealthy dietary pattern (including intake of large amounts of peroxidized polyunsaturated fatty acids PUFA in the diet, etc.).

According to another embodiment of the present invention, the therapeutic herbal composition exhibits anti-inflammatory activity because the therapeutic herbal composition effectively attenuates ROS formation, balances NADH/NAD ratio and downregulates inflammatory pathways. The therapeutic herbal composition of the present invention is also effective in controlling fatty liver formation.

According to yet other embodiments of the present invention, the therapeutic herbal composition of the present invention is effective in preventing reactive oxygen species (ROS) production in infected cells, thereby subsequently attenuating further degenerative pathways of inflammation in infected cells. Increased ROS production can be caused by various factors such as alcohol intake, drug intake, or unhealthy dietary patterns (including high intake of peroxidized polyunsaturated fatty acids PUFA in the diet, etc.). The therapeutic herbal composition is also effective in preventing depolarization of mitochondrial membranes in infected cells.

In one embodiment of the present invention, as established from in vitro studies, the therapeutic herbal composition of the present invention is effective against conditions such as alcohol, drugs and unhealthy dietary patterns by preventing nuclear apoptosis and nuclear fragmentation in infected cells Hepatotoxicity caused by other factors.

In yet other embodiments of the present invention, as established from preclinical studies, the therapeutic herbal composition of the present invention effectively inhibits serum enzymes, namely alanine aminotransferase (ALT), aspartate aminotransferase ( AST), alcohol dehydrogenase (ADH) and γ-glutamine transferase (GGT) concentrations increased.

According to another embodiment of the present invention, the therapeutic herbal composition helps to maintain Protects the liver from liver peroxidation. The same conclusion has been established through the in vitro and in vivo effect studies conducted by the inventors.

In one embodiment of the present invention, the therapeutic herbal composition of the present invention effectively reduces the concentration of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1), which is beneficial to reduce the Hepatic inflammation caused by a large amount of alcohol, intake of food containing a large amount of peroxidized polyunsaturated fatty acid (PUFA) or certain drugs, pathogenic infection, and alcoholic and non-alcoholic fatty liver disease.

According to still other embodiments of the present invention, the therapeutic herbal composition is effective to normalize and maintain lipid profile, thereby modulating lipid profile. In addition, the therapeutic herbal composition is effective in maintaining healthy cholesterol concentrations and healthy low-density lipoprotein to high-density lipoprotein (LDL/HDL) ratios. The composition is also effective in lowering elevated serum triglycerides. In addition, since the therapeutic herbal composition is effective in reducing elevated serum triglycerides and normalizing lipid profile, thereby it is also effective in controlling diseases such as diabetic retinopathy and age-related macular degeneration, because the therapeutic herbal composition Causes a rapid increase in macular pigment optical density (MPOD).

According to yet other embodiments of the present invention, since the therapeutic herbal composition exhibits lipid-lowering activity, it is also effective against liver toxicity that may be caused by alcohol consumption, drugs, and unhealthy dietary patterns.

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

According to another embodiment of the present invention, the botanical compounds curcuminoids and lutein exhibit synergistic activity in a therapeutic herbal composition. Furthermore, the curcuminoids and luteinoids present in the therapeutic herbal composition are highly bioavailable when compared to the bioavailability of higher doses of unformulated lutein and unformulated curcuminoids . In addition, these curcuminoids and lutein Present in amounts well below the recommended daily requirement.

According to another embodiment of the present invention, the therapeutic herbal composition may be taken/ingested as a supplement on a regular basis or before, during or after intake of fatty foods, drugs and/or alcohol.

The present invention also provides a method for the manufacture of a therapeutic herbal composition comprising curcuminoids and lutein, wherein the method comprises the following steps: a) using curcuminoid powder having a curcuminoid content in the range of 80 to 95% Lutein crystals/concentrate with a lutein content in the range of 70 to 80% and a zeaxanthin content of 4.5 to 9% and mix the curcumin powder/concentrate and lutein in a predetermined amount Crystals/concentrate to form a blend; the particle size of the untreated dry blend may fall within the range of D ₅₀ -20 μm to 50 μm and D ₉₀ -100 μm to 200 μm; b) excipients such as suitable emulsifiers and maltodextrin is added to the blend; c) purified water is added to the blend to make a suspension; d) the suspension is passed through a liquid colloid mill to form a homogeneous suspension; e) the suspension is Subjecting to further processing such as through a homogenizer or high shear particle wet mill to produce a micronized emulsion; f) further agitating the micronized emulsion at a speed of 25 rpm for 8 to 12 hours to raise the mass temperature to 25°C to 40°C, Preferably a temperature of 25°C to 30°C; g) subjecting the micronized emulsion to further processing such as concentration and/or drying to obtain the therapeutic herbal composition.

The method further comprises passing the dried therapeutic herbal composition through a suitable particle sieve to obtain a homogeneous finished product. The particle size of the treated therapeutic herbal composition may fall within the range of D ₅₀ -0.36 μm to 5 μm and D ₉₀ in the range of 0.60 μm to 10 μm. This particle size reduction facilitates high bioavailability of the therapeutic herbal composition.

In addition, the predetermined amounts of curcuminoid powder/concentrate and lutein crystals/concentrate can be such that the curcuminoid powder/concentrate is in the range of 24 to 49%, and the lutein crystals/concentrate is in the range of 1 to 49%. In the range of 14.5% and excipient in the range of 50 to 64%.

The excipients used are maltodextrin and emulsifier. In addition, some examples of emulsifiers that can be used are (but not limited to) modified starch, Gum Gum, Gum Arabic, Gum Acacia, methylcellulose, and sucrose fatty acid esters, etc. .

According to still other embodiments of the present invention, the final processed therapeutic herbal composition comprises curcuminoids in the range of 22.5% to 46.5%, lutein in the range of 0.90% to 11.25%, and lutein in the range of 0.06% to 1.12% zeaxanthin. It is apparent that the amount of zeaxanthin in the final composition constitutes approximately 6 to 9% of the total content of lutein in the final composition.

According to another embodiment of the present invention, the therapeutic herbal composition can be added to food and/or beverage products, formulated as dietary supplements, nutraceuticals and/or medicines.

According to another embodiment of the present invention, the therapeutic herbal composition can be used as a nutraceutical, medicine, dietary/nutritional supplement or as a therapeutic/health ingredient in various food and beverage products. Some examples of foods and beverages in which the therapeutic herbal composition can be used as a health ingredient include, but are not limited to, teas, infusions, fruit juices, beverages, milk and milk products, cereal products, alcoholic beverages, processed foods, and the like.

According to another embodiment of the present invention, the therapeutic herbal composition can also be formulated into a suitable dosage form selected from the group consisting of powder, paste, lozenge, syrup, infusion solution and/or capsule and the like.

In one embodiment of the present invention, the therapeutic herbal composition can also be added to any alcoholic beverage to counteract the adverse effects of alcohol consumption by people.

The following examples are intended to further illustrate certain preferred embodiments of the invention and are non-limiting in nature. Those skilled in the art will know, or can ascertain, using no more than routine experimentation, many equivalents to the specific materials and procedures described herein.

Example 1

In this example, a representative therapeutic herbal composition of the invention is described comprising various amounts by weight of curcuminoids, lutein and excipients. Table 1 shows the components and their amounts used in the therapeutic herbal compositions with different amounts of curcuminoids, lutein and excipients. Furthermore, the curcuminoids can be used in the form of curcuminoid powder or curcuminoid concentrate and lutein can be obtained from lutein crystals or lutein concentrate.

Example 2

The therapeutic herbal composition as detailed in Example 1 was manufactured by the method as described below: a curcuminoid powder/concentrate having a curcuminoid content of 80 to 95% and a lutein content of 70 to Lutein crystals at 80% and with a zeaxanthin content of 4.5 to 9% were mixed together with excipients such as modified starch emulsifier and maltodextrin to prepare a blend (100 g). The particle sizes of the dry blends thus obtained are D ₅₀ -20 μm and D ₉₀ -100 μm. This blend was then reconstituted with 333.4 ml RO purified water to make a suspension and further agitated until no lumps were present in the suspension. The suspension is then pre-emulsified in a liquid colloid mill or liquid wet mill to form a homogeneous emulsion. The pre-emulsion thus obtained is passed through a high pressure homogenizer/high shear particle wet mill to produce sub-micron sized emulsion droplets. The micronized emulsion is then further agitated at a speed of up to 25 rpm for 8 to 12 hours to raise the mass temperature to 25°C to 40°C, preferably 25°C to 30°C. This micronized emulsion was further spray dried at a chamber temperature not exceeding 110°C. The spray dried meal is then collected and subjected to dry milling using a micronizer or air assisted high shear dry milling. The ground particles are collected and passed through a suitable particle sieve and blended to obtain the final product. The particle size of the final powder is in the range of D ₅₀ −0.360 μm to 5 μm and D ₉₀ −0.60 μm to 10 μm.

100 gm of the therapeutic herbal composition of Example 1 manufactured by the above method contains the following active ingredients: curcuminoid content 22.5% to 46.5%; lutein content 0.90% to 11.25% and zeaxanthin content 0.06% to 1.12% . It is apparent that the amount of zeaxanthin in the final composition constitutes approximately 6 to 9% of the total content of lutein in the final composition.

Example 3

A therapeutic herbal composition as detailed in Example 1 was manufactured by the method as described below: 28.13 g of curcuminoid powder having a curcuminoid content of 80% and 14.10 g of lutein content of 80% and zeaxanthin Lutein crystals with a content of 5.72% were mixed together with 52.22 g of modified starch emulsifier and 10 g of maltodextrin to prepare a blend (100 g). The particle sizes of the dry blends thus obtained are D ₅₀ -40 μm and D ₉₀ -155 μm. This dry blend was then reconstituted with 333.4 ml RO purified water to make a suspension and further agitated until no lumps were present in the suspension. The suspension is then pre-emulsified in a liquid colloid mill or liquid wet mill to form a homogeneous emulsion. The pre-emulsion thus obtained is passed through a high pressure homogenizer/high shear particle wet mill to produce sub-micron sized emulsion droplets. The micronized emulsion is further agitated at a speed of up to 25 rpm for 8 to 12 hours to raise the mass temperature to 25°C to 40°C, preferably 25°C to 30°C. This micronized emulsion was further spray dried at a chamber temperature not exceeding 110°C. The spray dried meal is then collected and subjected to dry milling using a micronizer or air assisted high shear dry milling. The ground particles are collected and passed through a suitable particle sieve and blended to obtain the final product. The particle size of the final powder is D ₅₀ -2.76 μm and D ₉₀ -5.44 μm.

100 gm of the therapeutic herbal composition manufactured in Example 3 contained 22.5% curcuminoids, 11.25% lutein and 0.81% zeaxanthin.

Example 4

A therapeutic herbal composition as detailed in Example 1 was manufactured by the method as described below: 31.57 g of curcuminoid concentrate having a curcuminoid content of 95% and 3.85 g of lutein content of 78% and zeaxanthin Lutein concentrate with a content of 5.10% was mixed with 54.58 g of modified starch emulsifier and 10 g of maltodextrin to prepare a blend (100 g). The particle sizes of the dry blends thus obtained are D ₅₀ -50 μm and D ₉₀ -200 μm. This dry blend was then reconstituted with 333.4 ml RO purified water to make a suspension and further agitated until no lumps were present in the suspension. The suspension is then pre-emulsified in a liquid colloid mill or liquid wet mill to form a homogeneous emulsion. The pre-emulsion thus obtained is passed through a high pressure homogenizer/high shear particle wet mill to produce sub-micron sized emulsion droplets. The micronized emulsion is further agitated at a speed of up to 25 rpm for 8 to 12 hours to raise the mass temperature to 25°C to 40°C, preferably 25°C to 30°C. This micronized emulsion was further spray dried at a chamber temperature not exceeding 110°C. The spray dried meal is then collected and subjected to dry milling using a micronizer or air assisted high shear dry milling. The ground particles are collected and passed through a suitable particle sieve and blended to obtain the final product. The particle size of the final powder is D ₅₀ -0.6 μm and D ₉₀ -6.8 μm.

100 gm of the therapeutic herbal composition made in Example 4 contained 30% curcuminoids, 3% lutein and 0.20% zeaxanthin.

Example 5

A therapeutic herbal composition as detailed in Example 1 was manufactured by the method as described below: 48.95 g of curcuminoid powder having a curcuminoid content of 95% and 2.65 g of lutein content of 70% and zeaxanthin Lutein crystals with a content of 7.0% were mixed together with 38.40 g of modified starch emulsifier and 10 g of maltodextrin to prepare a blend (100 g). The particle sizes of the dry blends thus obtained are D ₅₀ -50 μm and D ₉₀ -200 μm. This dry blend was then reconstituted with 333.4 ml RO purified water to make a suspension and further agitated until no lumps were present in the suspension. The suspension is then pre-emulsified in a liquid colloid mill or liquid wet mill to form a homogeneous emulsion. The pre-emulsion thus obtained is passed through a high pressure homogenizer/high shear particle wet mill to produce sub-micron sized emulsion droplets. The micronized emulsion is further agitated at a speed of up to 25 rpm for 8 to 12 hours to raise the mass temperature to 25°C to 40°C, preferably 25°C to 30°C. This micronized emulsion was further spray dried at a chamber temperature not exceeding 110°C. The spray dried meal is then collected and subjected to dry milling using a micronizer or air assisted high shear dry milling. The ground particles are collected and passed through a suitable particle sieve and blended to obtain the final product. The particle size of the final powder is D ₅₀ -0.36 μm and D ₉₀ -2.2 μm.

100 gm of the therapeutic herbal composition manufactured in Example 5 contained 46.5% curcuminoids, 1.86% lutein and 0.18% zeaxanthin.

Example 6

The therapeutic herbal composition as detailed in Example 1 was manufactured by the method as described below: 48.95 g of curcuminoid concentrate having a curcuminoid content of 95% and 1.20 g of lutein content of 75% and zeaxanthin Lutein concentrate with a content of 5.65% was mixed with 54.58 g of modified starch emulsifier and 10 g of maltodextrin to prepare a blend (100 g). The particle sizes of the dry blends thus obtained are D ₅₀ -50 μm and D ₉₀ -200 μm. This dry blend was then reconstituted with 333.4 ml RO purified water to make a suspension and further agitated until no lumps were present in the suspension. The suspension is then pre-emulsified in a liquid colloid mill or liquid wet mill to form a homogeneous emulsion. The pre-emulsion thus obtained is passed through a high pressure homogenizer/high shear particle wet mill to produce sub-micron sized emulsion droplets. The micronized emulsion is further agitated at a speed of up to 25 rpm for 8 to 12 hours to raise the mass temperature to 25°C to 40°C, preferably 25°C to 30°C. This micronized emulsion was further spray dried at a chamber temperature not exceeding 110°C. The spray dried meal is then collected and subjected to dry milling using a micronizer or air assisted high shear dry milling. The ground particles are collected and passed through a suitable particle sieve and blended to obtain the final product. The particle size of the final powder is D ₅₀ -4.8 μm and D ₉₀ -9.9 μm.

100 gm of the therapeutic herbal composition manufactured in Example 6 contained 46.50% curcuminoids, 0.90% lutein and 0.068% zeaxanthin.

Example 7

In vitro studies evaluating the therapeutic effects of therapeutic herbal compositions on ethanol-induced and drug-induced oxidative stress on hepatocytes.

Purpose:

The main objective of the study was to investigate the effect of the therapeutic herbal composition of the present invention compared to silymarin on ethanol and acetaminophen (APAP) induced oxidative stress damage in human liver cancer (HepG2) cell lines. Since silymarin is widely used in the treatment of liver diseases, it was used to compare the efficacy of the novel therapeutic herbal composition of the present invention. In addition, since ethanol is a general anti-inflammatory agent and the liver is not considered a common target site of inflammation for this anti-inflammatory agent, the effect of the herbal composition on liver cells was studied and the anti-inflammatory agent was used in the form of ethanol.

Cell lines and media:

This work was performed in human hepatoma (HepG2) cells. The HepG2 cells were obtained from National Center for Cell Science, Pune, India. Cells were maintained in DMEM medium with 10% FBS, 1% glutamine and 100 U penicillin-streptomycin at 37°C in a 5% _CO2 atmosphere. Stock solutions were maintained in T- ^75cm tissue culture flasks.

experiment

Cytotoxicity studies

Evaluation of unprocessed curcuminoids (U1), unprocessed lutein (U2), unprocessed therapeutic herbal composition (U3) and processed curcuminoids (P1), processed lutein (P2) by MTT analysis ) and processed therapeutic herbal composition (P3), non-toxic concentrations of silymarin and its preventive effect against ethanol and acetaminophen (APAP)-induced cytotoxicity.

Sample preparation:

1. Unprocessed curcuminoid (U1) samples were prepared by using purified curcuminoid powder and mixing it with dry ingredients (ie emulsifier and maltodextrin) in predetermined amounts.

2. Prepare unprocessed lutein (U2) samples by using purified lutein concentrate and mixing it with dry ingredients (ie emulsifier and maltodextrin) in predetermined amounts.

3. Prepare untreated by mixing predetermined amount of curcuminoid powder, lutein with 6 to 9% zeaxanthin and further dry blending with predetermined amount of excipients such as emulsifier and maltodextrin Sample of therapeutic herbal composition (U3).

3. Prepare processed curcuminoid (P1 ) samples by using purified curcuminoid and mixing it with excipients (ie emulsifier and maltodextrin). This dry blend was then subjected to a treatment similar to that described for the therapeutic herbal composition in any of Examples 2-4 above.

4. Prepare processed lutein (P2) samples by using purified lutein and mixing it with excipients (ie emulsifier and maltodextrin). This dry blend was then subjected to a treatment similar to that described for the therapeutic herbal composition in any of Examples 2-4 above.

5. Prepare the treated therapeutic herbal composition sample (P3) as described in any one of Examples 2 to 6 above.

HepG2 cells were harvested and seeded in 96-well dishes at a density ( ^1x105 cells/well). To determine the toxicity of U1, U2, U3, P1, P2, P3 (1.95, 3.90, 7.81, 15.62, 31.25, 62.50, 125, 250, 500 and 1000 μg/ml) at 24 hours and perform MTT analysis. In order to evaluate the therapeutic effect of U1, U2, U3, P1, P2, P3 against ethanol and ethanol and APAP, different concentrations of U1, U2, U3, P1, P2, P3 (3.90, 7.81, 15.62, 31.25, 62.50 and 125 μg/ml) and silymarin (3.12 to 200 μg) for 1 h, and then exposed 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 prolonged for another 4 h. Then, 100 μl of DMSO was added and the absorbance was measured at 570 nm using a multimode plate reader.

Analysis of Lipid Peroxidation and Antioxidant Enzyme Activity

Measurement of malondialdehyde (MDA) (indicator of lipid peroxidation) concentration based on thiobarbituric acid reactive substances (TBARS) production, measurement of superoxide dismutase (SOD) activity according to a set of methods (Himedia and Sigma) and glutathione reductase (GSH). Samples were measured by standard spectrophotometry.

Cells were cultured with ethanol (100 mM) U1, U2, U3, P1, P2 and P3 and silymarin for 24 h. The total GSH content, SOD activity and lipid peroxidation in the therapeutic herbal composition were measured.

Cells were cultured with APAP (20mM), P3 and silymarin for 24h. The total GSH content, SOD activity and lipid peroxidation in the hepatoprotective composition were measured.

Measurement of intracellular reactive oxygen species (ROS)

ROS is measured using the non-fluorescent probe 2,7-diacetyldichlorofluorescein (DCFH-DH), which can penetrate into the intracellular matrix of cells, where the probe is oxidized to the fluorescent dichlorofluorescein by ROS prime (DCF). Briefly, aliquots of isolated cells (8 x ¹⁰⁶ cells/ml) were brought up to a final volume of 2 ml in normal phosphate buffered saline (pH 7.4). A 1 ml cell aliquot was removed, 1 μl of DCFH-DA (1 mg/mL) was added to the aliquot and incubated at 37° C. for 30 min in the dark. Images were taken on a fluorescent microscope (Nikon, Eclipse TS100, Japan) with a digital camera (Nikon 4500 coolpix, Japan).

With 62.50μg U1, U2, U3, P1, P2, P3 and 50μg silymarin HepG2 cells were treated for 1 hour and then treated with ethanol 100 mM for 24 hours. Intracellular ROS accumulation was measured using the fluorescent probe DCF-DA.

Measurement of Mitochondrial Membrane Potential (MMP)

Decreased MMPs are a sign of early apoptosis. In this study, differences in MMPs were detected by the fluorescent dye rhodamine 123 (Rh 123). HepG2 cells were cultured in 6-well dishes (1×10 ⁶ ) and harvested at the indicated end of treatment. After 24 hours of incubation with test compounds and ethanol or APAP, cells were incubated with Rh 123 (5 mmol/ml) for 15 minutes. Cells were then washed with PBS; fluorescence was observed under a fluorescent microscope using a blue filter (450 to 490 nm).

Double fluorescence and DAPI staining of HepG2 cells

HepG2 cells were grown in 35 mm cell culture dishes, treated with each candidate compound P3 (62.50 μg), silymarin (50 μg), ethanol 100 mM or APAP (20 mM) for 24 h, and 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 DAPI 100 mg mL was added to the cells, followed by fluorescence microscopy to assess morphological properties.

result:

A. Cytotoxic Effects of Untreated and Processed Components and Therapeutic Herbal Compositions in HepG2 Cells

Figure 1 (a, b, c) shows the cytotoxic effects of different concentrations of untreated components and therapeutic herbal compositions in HepG2 cells. Figure 1(d, e and f) shows the different concentrations of Cytotoxic effects of physiological components and treated therapeutic herbal compositions in HepG2 cells.

Figure 2 (a, b, c) shows the cytotoxic effects of different concentrations of untreated ingredients and therapeutic herbal compositions in ethanol-induced HepG2 cells. And Fig. 2 (d, e, f) shows the cytotoxic effects of different concentrations of the treated ingredients and therapeutic herbal composition in ethanol-induced HepG2 cells.

MTT analysis showed that in HepG2 cells, cell viability was unchanged at low concentrations ranging from 1.95 to 62.50 μg/ml. In addition, 3.9 to 62.50 μg/ml U1, U2, U3, P1, P2, P3 resulted in a significant increase in cell viability compared to ethanol. The maximum viability of the cells was observed when exposed to 62.50 μg/ml (Figure 2 (a, b, c, d, e and f)).

Therefore, U1, U2, U3, P1, P2, P3 at a concentration of 62.50 μg/ml were selected as the optimal protective concentration for all other studies.

B. Therapeutic Effects of Untreated and Treated Components and Therapeutic Herbal Compositions on Antioxidant Enzyme Activity in Ethanol-Induced HepG2 Cells

Thiobarbituric acid reactive substances (TBARS) assay was performed to investigate the ability of U1, U2, U3, P1, P2, P3 and silymarin to alter ethanol-induced lipid peroxidation in HepG2 cells (Fig. 3a). The results showed higher TBARS formation in ethanol-stimulated group compared to control cells. But the TBARS formation was reduced by treated cells, respectively.

The effects of U1, U2, U3, P1, P2, P3 and silymarin on ethanol-induced SOD activity and GSH concentration are shown in Figures 3(b) and 3(c). Compared with control cells, ethanol stimulation significantly decreased the concentration of non-enzyme antioxidant GSH and the activity of SOD in HepG2 cells. Interestingly, SOD and GSH increased in cells treated with U1, U2, U3, P1, P2, P3 and silymarin compared to the ethanol-stimulated group.

Furthermore, the effect of the treated therapeutic herbal composition (P3) was comparable to that of silymarin and the control group. Furthermore, the effect shown by P3 was superior to that shown by the untreated composition and individual components of curcumin (U1, P1) and lutein (U2, P2).

Therefore, it can be concluded from these results that the processed form (P3) of the therapeutic herbal composition of the present invention shows a synergistic activity of the individual components, namely curcuminoids and lutein (with 6 to 9% zeaxanthin) .

C. Cytotoxic effects of treated therapeutic herbal compositions compared to silymarin in HepG2 cells treated with acetaminophen (APAP)

Figures 4(a) and 4(b) show a comparison of the cytotoxic effect of the treated therapeutic herbal composition (P3) compared to silymarin in APAP-treated HepG2 cells.

In this study, APAP exposure caused significant cytotoxicity in HepG2 cells. However, pretreatment with the treated therapeutic herbal composition (P3) (62.50 μg) significantly prevented APAP-induced cytotoxicity in HepG2 cells; the results were comparable to those of silymarin (50 μg).

D. Therapeutic Effects of Treated Therapeutic Herbal Compositions Compared to Silymarin on Antioxidant Enzyme Activity on Acetaminophen (APAP)-Induced HepG2 Cells

It can be inferred from Figure 5(a) that the concentration of TBARS was increased in APAP-induced HepG2 cells when compared to control treatment. Furthermore, treatment with 62.50 μg of the treated therapeutic herbal composition (P3) before APAP induction significantly prevented APAP-mediated TBARS concentrations. These results are comparable to the effects shown by silymarin (50 μg).

Antioxidants serve as the primary defense against free radicals. APAP-induced HepG2 cells significantly decreased cellular antioxidant status due to excess ROS production. Instead, as in As can be seen in Figure 5(b) and (c), treatment with 62.50 μg of the treated therapeutic herbal composition (P3) and 50 μg of the given silymarin concentrate prior to induction of APAP significantly prevented APAP-induced antioxidant status in HepG2 cells (SOD and GSH concentration) loss.

E. Effect of Untreated and Treated Therapeutic Herbal Compositions Compared to Silymarin on Ethanol-Induced Intracellular ROS Production

It can be inferred from Figure 6 that the concentration of ROS was increased in HepG2 cells stimulated by ethanol alone compared to the untreated control group. In contrast, U1, U2, U3, P1, P2, P3 (62.50 μg/ml) and silymarin (50 μg/ml) significantly prevented ethanol-induced ROS production in HepG2 cells. The reduction in ROS production by treatment with 62.50 μg/mL of the treated therapeutic herbal composition (P3) was comparable to that of 50 μg/mL silymarin treatment.

F. Effect of Untreated and Treated Therapeutic Herbal Compositions Compared to Silymarin on Ethanol-Mediated MMP Reduction

Figure 7 depicts a significant decrease in mitochondrial membrane potential in cells exposed to ethanol 100 mM for 24 hours compared to controls. Depolarization of the mitochondrial membrane potential induced by disruption of the outer membrane resulted in a loss of the dye in the mitochondria and a decrease in intracellular fluorescence compared to controls. In contrast, pretreatment with 62.50 μg/mL of U1, U2, U3, P1, P2, and P3 and 50 μg/mL silymarin for 24 hours attenuated ethanol-induced mitochondrial membrane depolarization, as shown by an increase in fluorescence intensity.

The results indicate that the treated therapeutic herbal composition (P3) can significantly increase intracellular fluorescence, thereby indicating a positive effect on preventing the depolarization of the mitochondrial membrane potential of cells, and these results are comparable to those known for hepatoprotective The effect of silymarin.

G. Effect of Untreated and Treated Therapeutic Herbal Compositions Compared to Silymarin on Ethanol-Induced Nuclear Apoptosis

Figure 8 shows the fluorescence microscopy morphological changes in control and ethanol-induced HepG2 cells after staining with AO/EtBr. The figure is showing a photomicrograph showing the effect of treated therapeutic herbal composition (P3) 62.50 μg and silymarin 50 μg on the morphological changes of apoptosis induced by ethanol 100 mM in HepG2 cells. It can be seen that the treated therapeutic herbal composition (P3) inhibits apoptosis as stained by AO/EtBr staining in HepG2 cells, comparable to the effect shown by silymarin.

H. Effect of Untreated and Treated Therapeutic Herbal Compositions Compared to Silymarin on Ethanol-Induced Nuclear Fragmentation

As shown in Figure 9, treatment of HepG2 cells with 100 mM ethanol caused condensation and fragmentation of nuclei. It can be seen that both the treated therapeutic herbal composition (P3) and silymarin treatment before ethanol exposure reduced nuclear condensation and fragmentation due to their equally strong free radical scavenging properties.

I. Effect of Treated Therapeutic Herbal Compositions Compared to Silymarin on APAP-Induced Intracellular ROS Production

HepG2 cells treated with APAP 20 mM caused an increase in DCF fluorescence. Pretreatment of cells with treated therapeutic herbal composition (P3) 62.50 μg/ml decreased DCF fluorescence and it indicated that P3 significantly reduced APAP-induced free radical release compared to APAP alone treatment group (Figure 10).

J. Effect of Treated Therapeutic Herbal Compositions Compared to Silymarin on Acetaminophen (APAP)-Mediated MMP Reduction

As shown in Figure 11, after treatment with Rh-123, the mitochondrial membrane potential ( ΔΨm ) was measured by measuring the ratio of green fluorescence. APAP-treated cells were polarized and showed significant ΔΨm and reduced green fluorescence compared to controls. Pretreatment of cells with the treated therapeutic herbal composition (P3) increased green fluorescence, indicative of the depolarized state of the mitochondrial membrane, compared to cells treated with APAP alone. Furthermore, the treated therapeutic herbal composition (P3) and silymarin each showed similar effects.

K. Effects of Treated Therapeutic Herbal Compositions Compared to Silymarin on APAP-Induced Nuclear Apoptosis

Figure 12 shows photomicrographs showing the effect of treated therapeutic herbal composition (P3) 62.50 μg and silymarin 50 μg on APAP 20 mM induced apoptosis morphological changes in HepG2 cells. It can be concluded from Figure 12 that the treated therapeutic herbal composition (P3) inhibited apoptosis in HepG2 cells as stained by AO/EtBr staining and these results were comparable to the effect shown by silymarin.

L. Effect of Treated Therapeutic Herbal Compositions Compared to Silymarin on APAP-Induced Nuclear Fragmentation

As can be seen in Figure 13, HepG2 cells treated with 20 mM APAP caused nuclear condensation and fragmentation and morphological changes were observed in the cells. Pretreatment with the treated therapeutic herbal composition (P3) and silymarin prior to APAP exposure inhibited nuclear condensation and fragmentation compared to cells treated with APAP alone.

Example 8: In Vivo Studies

Effects of a therapeutic herbal composition against alcohol-induced liver injury in Weiss rats

Research purposes:

1. To monitor the effect of the therapeutic herbal composition (P3) on the changes of alcohol-induced liver injury in the initial and final body weight of experimental rats.

2. Evaluation of different doses of therapeutic herbal medicines by assessing lipid profile concentrations of triglycerides (TG), total cholesterol (TCH), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) Effect of composition (P3) on alcohol-induced hepatotoxicity.

3. Determine the activity of liver marker enzymes: aspartate aminotransferase (AST), alanine aminotransferase (ALT) and γ-glutamine transferase (GGT).

4. To assess the effect of different doses of the therapeutic herbal composition (P3) on alcohol-induced hepatotoxicity by assessing the lipid peroxidation markers malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE).

5. Assessment of markers of inflammation (tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) and interleukin 1 (IL-1 )) during alcohol-induced hepatotoxicity in rats.

Materials and methods:

animal-

Male Wess rats, weighing 130 to 150 g, were purchased from Biogen Laboratory Animal Facility, India. Animals were acclimatized with a 12h light:dark cycle at 25±2°C under standard ecobox conditions. Animals were fed standard rat chow and water ad libitum. Fasting for 18 to 24 hours before the experiment. The care and use of laboratory animals was carried out in accordance with the Good Laboratory Practices for Animal Experiments in accordance with the Indian CPCSEA Council Directive (MCAS/IAEC/2019/6/9/2/2019).

Experimental design and treatment schedule:

The experimental male Weiss rats were divided into six groups, and each group contained six animals. The total experimental period of 28 days and the doses of ethanol, silymarin and the treated therapeutic herbal composition P3 were analyzed as follows:

The control group and the ethanol group received 0.9% saline via intragastric injection for 28 days, respectively. To examine the therapeutic properties, silymarin and a therapeutic herbal composition (P3) were administered to rats at a dose of 200 mg/Kg body weight 30 min before ethanol administration for 28 days.

Composition of animal feed:

Protein - 17.7%, Fat - 4.2%, Carbohydrate - 50.5%, Fiber - 3.4%, Mineral - 6.7% and Vitamin - 1.7%.

On day 28 of the study, all animals were anesthetized with chloroform, and blood samples were collected intravenously from control and experimental rats. Blood samples were centrifuged at 3000 rpm for 30 min, and serum samples were stored in a -20°C freezer until analysis. The livers of sacrificed animals were also immediately removed, rinsed appropriately in ice-cold saline, blotted dry and weighed to record organ weights. A portion of the liver tissue was then immediately immersed in an appropriate fixative for further histopathological studies. The remainder of the liver was saved for biochemical analysis.

Measurement of liver weight and body weight

The body weight of all animals at the end of the treatment period was recorded, together with their liver weight on the day of sacrifice, to determine liver weight and final body weight of animals in each group.

Preparation of tissue homogenate

A 10% w/v liver tissue homogenate was prepared in ice-cold phosphate buffered saline (PBS) (pH 7.4) containing 1 mM EDTA 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 analysis of tissue oxidative stress markers.

biochemical analysis

Protein content was estimated by the method of Lowry et al. (1951). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamine transferase (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) analysis.

Liver 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 the cytosolic fraction in a final volume of 1 ml. Enzyme activity was measured at 340 nm and calculated as nmol NADH formation/min/mg protein using a molar extinction coefficient of 6.22 x 106 M-1 cm-1.

Cytokines Assessment

Pro-inflammatory interleukins, namely tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) and interleukin 1 (IL-1), were also assessed using commercially available ELISA kits, and skin grams per milliliter (pg/mL) (Biovision, Milpitas, CA, USA).

Histopathological studies

A portion of rat liver tissue from control and treated animals was directly fixed in 10% buffered formalin and embedded in paraffin after periodic washing with saline and dehydration. After all procedures, 3 to 5 μm thick sections were then prepared from the tissue blocks using a microtome. The tissue sections were then stained with hematoxylin and eosin (H&E). The tissue sections are observed under a microscope and corresponding images are captured using a digital camera attached to them.

Statistical analysis

Each experiment was repeated at least three times. Data are expressed as mean ± S.E. After determining the homogeneity of variance between treatment groups, the significance of the means of the different parameters between these treatment groups was analyzed using a one-way post-hoc test (Tukey HSD test) of analysis of variance (ANOVA). Statistical tests were performed using SPSS software version 20.0. Values of p<0.05 were considered statistically significant.

result:

A. Effects of Treated Therapeutic Herbal Compositions on General Observed Body Weight

Table 2 shows the water and food intake in control and experimental rats. There was no difference in food and water consumption between control and experimental rats throughout the study period.

Tables 3 and 3a show the effects of ethanol and the treated therapeutic herbal composition of the present invention (P3) on body weight, body weight gain and growth rate and liver weight in control and experimental groups of rats, respectively. Body weight decreased and liver weight increased significantly ( P < 0.05) in ethanol-induced rats compared to untreated control (group 1) rats, while liver weight increased significantly (P < 0.05) in ethanol-induced rats when compared to group 2 rats It appeared to be close to normal in the herbal composition (P3) treated rats (groups 4 to 7). Body weight, body weight gain and growth rate were significantly reduced ( p <0.05) in group 2 rats when compared to group 1 rats. Treatment with the therapeutic herbal composition of the invention (P3) with varying amounts of curcuminoids and lutein (in addition with 6 to 9% zeaxanthin) also showed efficacy against body weight loss induced by ethanol treatment of rats. Protective and comparable to the effect shown by silymarin.

*Values are given as mean SD of each group.

*Values are given as mean SD of each group.

*Values are given as mean SD of each group.

B. The effect of the processed therapeutic herbal composition (P3) of the present invention on changes in liver function marker enzymes

Table 4 indicates the changes in liver marker enzyme activity in control and experimental rats. The administration of ethanol significantly increased ( p <0.05) the concentrations of AST, ALT and GGT, respectively. Finally, the treated therapeutic herbal composition of the present invention (P3) with an amount of 4 mg to 52 mg of curcuminoids and 1.05 mg to 2 mg of lutein at a dose of 200 mg/Kg compared to ethanol-treated rats. Treatment (Groups 4 to 7) significantly ( p <0.05) reduced elevated concentrations of AST, ALT and GGT. Furthermore, the results (groups 4 to 7) shown by the treated therapeutic herbal composition of the invention (P3) with curcuminoids in amounts of 4 mg to 52 mg and lutein in amounts of 1.05 mg to 2 mg were comparable to those of silymarin.

Table 4: Effects of the therapeutic herbal composition (P3) on the changes of alcohol-induced liver function marker enzymes in control and experimental rats in each group (n=6).

*Values are given as mean SD of each group.

Ethanol consumption enhances the NADH/NADP ratio in hepatocytes, which causes disruption of β-oxidation of fatty acids in mitochondria, leading to steatosis. Alcohol also increases lipid transport from the small intestine to the liver, which results in increased mobilization of fatty acids from adipose tissue and fatty acid absorption by the liver (1). This causes damage to the cell membrane of the liver cells, which leads to a decrease in the concentration of transaminases (alanine aminotransferase (ALT) and AST (aspartate aminotransferase)) in the bloodstream. Gamma-glutamine transferase (GGT) plays a key role in maintaining intracellular oxidative stress homeostasis, which protects cells from oxidative damage. Gamma-glutamine transferase is present in the cell membrane and is released in circulation when the cell membrane is damaged.

The findings of the present invention show that ethanol administration triggers a significant increase in serum AST, ALT and GGT enzyme activities compared to the control group. These increases are due to liver inflammation caused by alcoholism. Administration of the treated therapeutic herbal composition of the present invention (P3) with varying amounts of its components, namely curcuminoids and lutein (in addition with 6 to 9% zeaxanthin) was effective in alleviating the Increased serum enzyme concentrations in rats resulted in a subsequent return to normal, which was comparable to that of control animals.

C. Effect of the treated therapeutic herbal composition of the invention (P3) on ethanol-induced changes in lipid profile.

Table 5 shows that by assessing the lipid profiles in control and experimental rats, namely TG, TCH, LDL-C concentration and HDL-C concentration, comparing their components with different amounts, namely curcuminoids and lutein (in addition to Effect of a treated therapeutic herbal composition (P3) from 6 to 9% zeaxanthin) on ethanol-induced hepatotoxicity.

As can be inferred from Table 5, treatment with ethanol also disrupted the body's lipid profile homeostasis, resulting in an increase in TG, TCH and LDL-C concentrations, respectively, along with a concomitant decrease in HDL-C concentration (p<0.05). Co-treatment with the treated therapeutic herbal composition of the present invention (P3) significantly reversed the above changes in TG, TCH and LDL-C, HDL-C concentrations to near normal values (p<0.05). During ethanol metabolism, a large amount of reduced nicotinamide-adenine dinucleotide (NADH) is produced, thereby inhibiting the Krebs cycle and fatty acid oxidation, which is beneficial to hepatic steatosis and serum hyperlipidemia (2).

In addition, the ethanol group also showed an increase in TCH, TG, and LDL concentrations along with a simultaneous decrease in HDL concentrations. However, treatment with the treated therapeutic herbal composition (P3) attenuated the above changes in lipid profile concentrations. Therefore, the therapeutic herbal composition (P3) of the present invention is effective in maintaining this lipid profile. Furthermore, the composition is effective in maintaining healthy cholesterol concentrations and healthy LDL/HDL ratios and reducing elevated serum triglyceride concentrations.

The hypocholesterolemic activity of animals treated with the treated therapeutic herbal composition (P3) can be attributed to increased hepatic clearance of cholesterol by elevated serum HDL concentrations, or to reduction of the cholesterol biosynthetic enzyme HMG-CoA Enzyme (3) downregulation.

*Values are given as mean SD of each group.

D. The effect of the processed therapeutic herbal composition (P3) of the present invention on the changes of liver ADH, MDA and 4-HNE induced by alcohol

Table 6 shows the effect of the treated therapeutic herbal composition of the invention (P3) with different amounts of its components, namely curcuminoids and lutein (in addition with 6 to 9% zeaxanthin) on ethanol-induced hepatic ADH , Comparison of the effects of MDA and 4-HNE concentration changes. Compared with the control group, the ethanol-treated group (group 2) showed a significant increase in ADH, MDA and 4-HNE concentrations. Increased concentrations of ADH, MDA and 4-HNE in the ethanol control group indicated the presence of a lipid profile of hepatocytes due to the toxic effects of ethanol.

Co-treatment with the treated therapeutic herbal composition of the invention (P3) at a dose of 200 mg/Kg with curcuminoids in amounts of 4 mg to 52 mg and lutein in amounts of 1.05 mg to 2 mg compared to ethanol-treated rats (Groups 4 to 7) significantly ( p <0.05) reduced elevated ADH, MDA and 4-HNE concentrations.

*Values are given as mean SD of each group.

E. Effect of the Treated Therapeutic Herbal Composition (P3) of the Invention on Alcohol-Induced Changes in Cytokinin Concentrations

Table 7 indicates the effect of the treated therapeutic composition of the invention (P3) on ethanol-induced changes in cytokine concentrations. It can be seen that compared with the control group 1, the administration of ethanol led to a significant increase in serum TNF-α, IL-6 and IL-1 concentrations (p<0.001). Table 7 shows that in ethanol-treated group 2, serum TNF-α, IL-6 and IL-1 concentrations were increased, respectively. As seen in the results for Groups 4 to 7, co-treatment with the treated therapeutic herbal composition (P3) caused a significant reduction in serum TNF-α, IL-6 and IL-1 concentrations.

Table 7: Effects of the therapeutic herbal composition (P3) of the present invention on ethanol-induced changes in cytokine concentrations

*Values are given as mean SD of each group.

F. Histopathological analysis of liver tissue

The histological features shown in Figure 14 are indicative of normal hepatic lobular architecture and cellularity of the liver in control rats. There were no pathological changes in healthy control livers. In ethanol-induced rats (ie, Group 2), the extent of liver injury was assessed by histological analysis after 4 weeks of continuous ingestion of 2.4 gm/kg bw ethanol. For example, sinusoidal dilatation, extrusion, and cord atrophy were observed in centrilobular hepatocytes, possibly indicative of hepatocyte regeneration; a slight infiltration of monocytes and necrotic cells was observed around the central vein. Cavitary changes and possible steatosis were observed in the centrilobular region. Briefly, hepatocyte swelling, watery degeneration, and vacuolation became evident in the centrilobular region, reflecting early biochemical and pathological changes resulting from alcoholic liver injury.

Rats in ethanol-treated group 2 showed inflammation around the central vein with vacuolar degeneration and necrosis. In groups 3, 4 and 5, liver tissue showed less vacuolar degeneration and less inflammatory response around the central vein. Groups 6 and 7 liver tissues showed almost normal structure. Therefore, the present invention The treated therapeutic herbal composition (P3) significantly improved ethanol-induced histopathological changes, especially in the compositions of the present invention (P3) with curcuminoids in amounts from 4 mg to 52 mg and lutein in amounts from 1.05 mg to 2 mg (Groups 4 to 7), which significantly reduced steatosis in the liver and showed significant improvement compared to the ethanol-treated group.

conclusion of the study

In conclusion, in vivo studies demonstrate that the treated therapeutic herbal composition (P3) of the present invention effectively normalizes and maintains the lipid profile. Furthermore, the composition is effective in maintaining healthy cholesterol concentrations and healthy LDL/HDL ratios and is effective in reducing elevated serum triglyceride concentrations. Furthermore, the therapeutic herbal composition of the present invention is effective in normalizing and maintaining serum enzymes (ie AST, ALT, ADH and GGT) concentrations. Studies evaluating lipid peroxidation markers MDA and 4-HNE also showed that the therapeutic herbal composition (P3) effectively reduces lipid peroxidation that can be caused by various factors such as unhealthy dietary pattern, high consumption of PUFA and alcohol consumption. Furthermore, the therapeutic herbal composition (P3) of the present invention was found to be effective in reducing serum concentrations of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1), which are markers of inflammatory response. It is believed that these serum pro-inflammatory cytokines (TNF-α, IL-6 and IL- 1) Significantly increased. Furthermore, the therapeutic herbal composition favorably ameliorate ethanol-induced hepatotoxicity by protecting the liver status in terms of its antioxidant status, reducing lipid profile, liver enzyme marker concentration and anti-inflammatory activity and against chronic alcohol exposure.

It is also concluded from the results that the therapeutic herbal composition of the present invention has lipid-lowering and anti-inflammatory activities. Some of the causative factors for abnormal lipid profile and inflammation in the body can be unhealthy eating patterns, high alcohol intake, intake of foods with high peroxidized polyunsaturated fatty acid (PUFA) content, or due to taking certain medications. Inflammation in the body can also be caused by pathogenic infection, the pathogenic infection Infections are caused by infectious agents/pathogens such as viruses (including viruses such as coronavirus disease (COVID-19) and other viruses that cause infection) and bacteria.

Histopathological analysis of liver tissue indicated that the treated therapeutic herbal composition of the present invention (P3) significantly improved the changes induced by ethanol, especially in the presence of curcuminoids in amounts of 4 mg to 52 mg and lutein in amounts of 1.05 mg to 2 mg. In the composition (P3) of the invention.

Example 9: Clinical Research

Randomized, open-label, single-center, one-period, single-dose, three-arm bioavailability study of a therapeutic herbal composition in healthy human adult male subjects under fasting conditions

Research purposes:

Primary (pharmacokinetic) purpose:

The bioavailability of curcuminoids and lutein in the therapeutic herbal composition of the present invention was determined compared to the bioavailability of unformulated curcuminoids and unformulated lutein.

Secondary (security) purpose:

The secondary objective was to monitor the safety and tolerability of a single dose of the therapeutic herbal composition of the present invention in 9 healthy human adult male subjects under fasting conditions.

Inclusion criteria

(a) Subjects must be healthy human male subjects.

(b) Must be between 18 and 65 years old (inclusive) and weigh at least 50kg.

(c) be prepared to provide written informed consent and should be willing to be available throughout the course of the study and should Subjects following the guidelines mentioned in the protocol.

(d) Subjects with no evidence of underlying disease during pre-study screening. Subjects must be healthy as determined by medical history and physical examination, ECG, chest X-ray (PA view), and laboratory tests performed within 14 days prior to the start of the study.

(e) Subjects whose screening laboratory values are within normal limits or considered clinically significant by the physician/principal investigator.

Exclusion criteria:

1. Subjects who are allergic to turmeric or its extracts or curcuminoids or lutein or any food or other drugs.

2. Subjects with resting hypotension (BP<90/60) or hypertension (BP>140/90) and pulse rate below 50/min and over 100/min.

3. Suffering or past medical history or significant cardiovascular, lung, liver, kidney, blood, gastrointestinal, endocrine, immune, skin, nerve, musculoskeletal or mental diseases, or hospitalization or surgery within the last 4 weeks before admission subject.

4. Subjects with a history of MI, stroke, peripheral artery disease, GI hemorrhage, liver injury, asthma, kidney injury, epilepsy and intracranial hemorrhage.

5. Subjects who have taken over-the-counter or prescription drugs (including any enzyme-modifying drugs) within the last 14 days before the study.

6. Subjects with a history of alcoholism, drug abuse or smoking.

7. Subjects with high sensitivity to heparin.

8. Subjects who participated in any other clinical research in the past three months.

9. With clinically significant abnormal laboratory values/abnormal ECG/abnormal chest radiograph (PA view) of subjects.

10. Subjects who have difficulties in donating blood.

11. Subjects with a history of dysphagia.

12. Subjects with veins not suitable for repeated venipuncture.

outcome assessment

Bioavailability of curcuminoids and lutein in respective formulations

research program

A. Design - Randomized, Pilot, Double-Blind, Single-Center, One-Period, Single-Dose, Three-Arm Bioavailability Study

Research Processing Assignment

Group I: 3 subjects, a therapeutic herbal composition comprising 120 mg of curcuminoids and 20 mg of lutein

Group II: 3 subjects, a therapeutic herbal composition comprising 30 mg of curcuminoids and 5 mg of lutein

Group III: 3 subjects, reference unformulated curcuminoids containing 500 mg curcuminoids and 40 mg unformulated lutein

B. Number of subjects - 9 healthy male volunteers

C. Randomization (Assignment to Treatment Order) - Randomization is performed by appropriate statistical software (SAS version 9.13 or higher) to achieve an equal number of subjects in all arms. The operations team cannot A randomization schedule was obtained to keep their peers blinded to treatment assignment.

D. Overall research plan

Subjects were asked to visit the site after ethics committee approval was obtained. After obtaining written informed consent from healthy subjects, subjects were asked about their medical history and researchers performed a physical examination. Blood samples were taken from each subject for analysis of hematology and biochemical analyses. X-rays and ECGs are performed. Subjects were enrolled in the study after meeting all IC/EC criteria.

Subjects entered the facility 12 hours prior to dosing. The subjects were provided with a turmeric-free dinner. Perform physical examination and vital signs.

On the day of administration, physical examination and vital signs were performed and recorded. A 5 ml predose sample was collected and centrifuged for pharmacokinetic analysis. All subjects were dosed. According to the randomization schedule, 1 gm of the investigational product (IP) in the sachet was mixed with 240 ml of water and the subjects were asked to drink the water with the IP. After administration, the time points of blood collection were: 0.50, 1, 2, 3, 4, 6 and 24 hours for pharmacokinetic analysis. Vital signs were recorded at these time points: 00.15, 02.00, 04.00, 06.00, 09.00 and 12.00 hours after dosing. Physical examinations were performed at these time points: 1, 2, 6, 12 hours after dosing. At 04.00, 08.00 and 13.00 hours after dosing (ie lunch, snack and dinner respectively), the subjects were provided with food without turmeric. Adverse events (if any) were recorded.

Physical examination and vital signs were performed at the time of examination (24 hours after dosing). Blood samples were collected 24 hours after dosing for pharmacokinetic analysis. Adverse events (if any) were recorded. Blood samples were collected for safety analysis. The subjects were discharged from the facility after completion of all post-dose procedures.

E. Pharmacokinetic Analysis

Plasma samples were analyzed using a fit-for-purpose LC-MS method to quantify total curcuminoids (curcumin, demethoxycurcumin and bisdemethoxycurcumin) and lutein (in addition to 6 to 9% corn Xanthophyll). Pharmacokinetic parameters are calculated using standard analytical tools. The relative bioavailability of the components from the therapeutic herbal composition was compared and the AUC _(0-24h) from the same assay was reported for unformulated curcuminoids and unformulated lutein.

F. Statistical Analysis

Statistical analysis was performed and AUC was calculated using InStat software (GraphPad Prism, USA). Two-tailed ANOVA was used to analyze total plasma curcuminoid levels between and within groups, and a "p" value of less than 0.05 was calculated as statistical significance for the study.

G. Results and Discussion

All 9 subjects with no medical history were screened. The mean age of the subjects in Group I included in the study was 28.4, Group B was 25.9 and Group C was 23.0 with a mean of 25.76. The average height of the volunteers in Group I was 162.00, Group II was 167.00, Group III was 163.00 and the mean was 164.0. At the baseline visit, the mean weight was 65.0 for Group I, 75.80 for Group II and 63.0 for Group III with a mean of 69.6 and a mean BMI of 25.60 kg/ ^m2 .

Safety: During the study, vital signs (such as temperature, systolic and diastolic blood pressure, pulse rate and respiration rate) were measured and recorded at all time points before and after dosing. This clearly demonstrated that all subjects had no clinically significant value and therefore all investigational products (therapeutic herbal compositions of the present invention) were completely safe for administration. No adverse events were observed.

Therefore, it can be concluded that the therapeutic herbal composition of the present invention in 1 g sachet formulation is completely safe for human oral administration.

Bioavailability:

A. Curcuminoids

As can be seen in Table 8, in all groups, the median time to reach peak plasma curcuminoid concentration (Tmax) after oral administration of the product was found to be 2 hours.

In group I, exposures (C _max and AUC _last ) were found to be 20.70 and 61.93, respectively, while in group II, exposures (C _max and AUC _last ) were found to be 2.51 and 45.48, respectively, while in group III, exposures ( C _max and AUC _last ) were 8.98 and 58.83, respectively. The mean plasma concentration-time profiles of curcuminoids are presented in FIG. 15 .

Therefore, it can be concluded that the curcuminoids present in the treated therapeutic herbal composition are highly bioavailable upon ingestion when compared to the bioavailability of unformulated curcuminoids at higher doses.

B. Lutein

As can be seen in Table 9, in all groups the median time to reach peak plasma lutein concentration (Tmax) after oral administration of the product was found to be 6 hours.

In group I, exposures (C _max and AUC _last ) were found to be 55.88 and 424.87, respectively, while in group II, exposures (C _max and AUC _last ) were found to be 35.18 and 287.70, respectively, while in group III, exposures ( C _max and AUC _last ) were 52.27 and 365.07, respectively. The mean plasma concentration-time profiles of lutein are presented in FIG. 16 .

Therefore, it can be concluded that the lutein present in the treated therapeutic herbal composition is highly bioavailable upon ingestion when compared to the bioavailability of unformulated lutein at higher doses.

While particular embodiments of the therapeutic herbal compositions 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 without departing from the spirit and scope of the invention. Accordingly, it is intended to cover in the appended claims such changes and modifications as are within the scope of this invention.

references:

(1) Shukla I, Azmi L, Gupta SS, Upreti DK, Rao CV. (2018) Melioration of anti-hepatotoxic effect by Lichen raniferinus against alcohol induced liver damage in rats. J Ayurveda Integr Med. 2018 Jan 29. pii: S0975 -9476(17) 30047-5.

(2) 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 .

(3) 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 (20)

A therapeutic herbal composition comprising powdered curcuminoids present in the range of 24 to 49%, lutein crystals present in the range of 1 to 14.5%, and excipients present in the range of 50 to 64%. Forming agent, wherein the curcuminoid powder has a curcuminoid content of 80 to 95%, and the lutein crystal has a lutein content of 70 to 80% and a zeaxanthin content of 4.5 to 9%. A therapeutic herbal composition comprising a concentrate of curcuminoids present in the range of 24 to 49% and a concentrate of lutein present in the range of 1 to 14.5% and a concentrate of lutein present in the range of 50 to 64% An excipient for an excipient, wherein the curcuminoid concentrate has a curcuminoid content of 80 to 95%, and the lutein concentrate has a lutein content of 70 to 80% and a zeaxanthin content of 4.5 to 9%. The therapeutic herbal composition according to claim 1 or 2, wherein the composition is effective in regulating blood lipid profiles and regulating inflammatory effects, which may be caused by various factors. The therapeutic herbal composition as claimed in item 3, wherein the factors include but are not limited to unhealthy eating patterns, intake of a large amount of alcohol, intake of foods containing a large amount of peroxidized polyunsaturated fatty acid (peroxidized Polyunsaturated fatty acid; PUFA) content or due to Taking certain medications and pathogenic infections due to pathogens, including viruses and bacteria. The therapeutic herbal composition as claimed in item 3, wherein the pathogen includes coronavirus COVID-19. The therapeutic herbal composition according to claim 3, wherein the composition is effective in at least one of the following diseases, namely: (a) inhibiting oxidative stress damage in infected cells; (b) increasing The content of non-enzyme antioxidant glutathione reductase (Glutathione reductase; GSH) and superoxide dismutase (superoxide dismutase; SOD); (c) prevention of reactive oxygen species (reactive oxygen species; ROS) in these infected cells ) production; (d) preventing nuclear apoptosis, nuclear fragmentation and preventing mitochondrial membrane depolarization in the infected cells. The therapeutic herbal composition according to claim 3, wherein the composition effectively reduces the production of Thiobarbituric acid reactive species (Thiobarbituric acid reactive species; TBARS) Malondialdehyde (MDA) in the infected cells. The therapeutic herbal composition according to claim 1 or 2, wherein the composition is effective in normalizing and maintaining lipid profile, maintaining healthy cholesterol levels and healthy LDL/HDL ratio and reducing high serum triglyceride levels, thereby effectively controlling disease. The therapeutic herbal composition according to claim 8, wherein the diseases include but not limited to diabetic retinopathy and age-related macular degeneration. The therapeutic herbal composition as claimed in item 1 or 2, wherein the composition effectively reduces the content of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1), which is beneficial to reduce the Ingestion of a large amount of alcohol, intake of food containing a large amount of peroxidized polyunsaturated fatty acid (PUFA) or due to taking certain drugs, due to alcoholic and non-alcoholic fatty liver disease and liver inflammation caused by pathogenic infection. The therapeutic herbal composition according to claim 1 or 2, wherein the curcuminoids and lutein present in the therapeutic herbal composition exhibit synergistic activity and when compared to higher doses of unformulated lutein and unformulated The bioavailability of curcuminoids showed high bioavailability. The therapeutic herbal composition according to claim 1 or 2, wherein the composition can be taken/ingested regularly as a supplement or before, during or after intake of high-fat food, drugs and/or alcohol. The therapeutic herbal composition as claimed in item 1 or 2, wherein the curcuminoid powder/concentrate can be obtained/extracted and purified from the plant turmeric ( Curcuma longa ) and the lutein crystals/concentrate can be obtained from marigold The plant Marigold ( Tagetes erecta ) or any other suitable plant source is obtained/extracted and purified. The therapeutic herbal composition as claimed in claim 1 or 2, wherein the composition is formulated and used as a nutritional product, a medicine, a dietary/nutritional supplement or as a therapeutic/healthy ingredient in various food and beverage products tea, infusion , fruit juices, beverages, milk and milk products, cereal products, alcoholic beverages and processed foods. A method for the manufacture of a therapeutic herbal composition according to any one of claims 1 to 14, wherein the method comprises: a) using a curcuminoid powder/concentrate with a curcuminoid content in the range of 80 to 95% Lutein crystals/concentrates with lutein content in the range of 70 to 80% and zeaxanthin content in the range of 4.5 to 9% and mixed with the curcumin powder/concentrate and lutein crystals/ Concentrate to form a blend; the particle size of the untreated dry blend may fall within the range of D ₅₀ -20 μm to 50 μm and D ₉₀ -100 μm to 200 μm; b) adding excipients to the blend; c ) adding purified water to the blend to make a suspension; d) passing the suspension through a liquid colloid mill to form a homogeneous suspension; e) subjecting the suspension to further processing, wherein the processing includes passing through a homogenizer or High shear particle wet mill to produce micronized emulsion; f) further agitating the micronized emulsion at a speed of 25 rpm for 8 to 12 hours to raise mass temperature to 25°C to 40°C; g) subjecting the micronized emulsion to further processing. The method of claim 15, wherein the excipient may include suitable emulsifiers and maltodextrin, and wherein the further processing of the micronized emulsion includes concentration and/or drying to obtain the therapeutic herbal composition. The method of claim 15 or 16, wherein the curcuminoid powder is present in the range of 24 to 49%, and the lutein crystal system is in the range of 1% to 14.5% and the excipients are in the range of 50 to 64%. within the range. The method of claim 15 or 16, wherein the curcuminoid concentrate exists in the range of 24 to 49%, and the lutein concentrate is in the range of 1% to 14.5%, and the excipients are in In the range of 50 to 64%. The method of claim 15 or 16, wherein the therapeutic herbal composition is further passed through a suitable particle sieve to obtain a uniform finished product and wherein the particle size of the treated herbal composition is in the range of _D50 -0.36 μm to 5 μm Inner and D ₉₀ are in the range of 0.60 μm to 10 μm. The method of claim 15 or 16, wherein the curcuminoid powder/concentrate can be obtained/extracted and purified from the plant Curcuma longa and the lutein crystals/concentrate can be obtained from the marigold plant Marigold or any other suitable Obtained/extracted and purified from plant sources.