DESC:FIELD OF THE INVENTION

The present invention relates to a composition comprising omega-3 fatty acids in synergy with one or more natural compounds and a surfactant, wherein the synergistic interaction among the compounds increases cardioprotective activity by promoting the reduction of triglyceride levels in blood plasma. The invention further relates to a nanoemulsion formulation of omega-3 fatty acids, with one or more natural compounds and a surfactant designed to enhance bioavailability, thereby improving the absorption and efficacy of the active compounds.

BACKGROUND OF THE INVENTION AND RELATED PRIOR ARTS

Omega-3 fatty acids
Omega-3 polyunsaturated fatty acids (PUFAs) including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are essential fatty acids which need to be supplied externally through diet by animals having various physiological functions. (Hindawi, Mediators of Inflammation Volume 2021, Article ID 8879227, https://doi.org/10.1155/2021/8879227).

EPA and DHA are considered to be crucial for proper fetal development and maintaining health throughout aging. Beyond their physiological functions, EPA and DHA serve as biochemical precursors to various metabolites, which function as powerful lipid mediators. These metabolites are widely recognized in scientific literature for their potential therapeutic or preventive benefits in addressing a variety of health conditions. The main dietary sources of EPA and DHA come from marine-based products, with fish and fish-oil supplements being the primary contributors of these biologically active compounds. (2012 American Society for Nutrition. Adv. Nutr. 3: 1–7, 2012; doi:10.3945/an.111.000893.)

Oxidative stress and chronic inflammation are recognized as key contributors to the development of non-communicable diseases, including cardiovascular disease, Poly unsaturated fatty acids (PUFAs) have been shown to modulate antioxidant signaling pathways and inflammatory responses, thereby influencing systemic physiological functions. Higher intake of DHA and EPA, is consistently associated with a reduced incidence of chronic-inflammation related diseases, including cardiovascular disease, likely mediated through reductions in blood cholesterol levels. (Nutrients 2021, 13, 2421. https://doi.org/10.3390/nu13072421).

Over the past decade, elevated triglyceride (TG) levels have been identified as independent risk factors for coronary heart disease (CHD), regardless of other lipid profile abnormalities. Clinical reviews evaluating the impact of long-chain omega-3 fatty acids on serum lipids and lipoproteins have demonstrated that the daily intake of approximately 4 grams of long-chain omega-3 fatty acids results in a reduction in serum TG levels by approximately 25-30%, irrespective of baseline TG concentrations. (doi:10.1111/j.1753-4887.2010.00272.x Nutrition Reviews® Vol. 68(3):155–167).

PUFAs, particularly DHA, shows potential in the management of depressive symptoms in patients with cognitive impairment. Clinical evidence suggests that such supplementation, especially in individuals with mild cognitive impairment (MCI), may provide therapeutic benefit. (Healthcare 2024, 12, 536. https://doi.org/10.3390/healthcare12050536).

DHA is found in high concentrations in the brain cell membranes and supports nervous system function, suggesting a possible protective role in Alzheimer’s disease (AD), a serious, progressive condition with no known cure and only a few treatment choices. Inflammation, triggered by immune cells in the brain like microglia, is believed to be a key factor in AD. These cells release inflammatory molecules such as IL-1 ß, IL-6, and TNFa which may result in the dysfunction of neurons. Studies have shown AD patients who received EPA and DHA supplements showed a decrease in the release of these inflammatory markers.

PUFAs are beneficial in the treatment of non-alcoholic fatty liver disease (NAFLD) and are linked to a reduced risk of colorectal cancer (CRC) in cases of high levels of FOXP3+ T cell infiltration suggesting cancer immunopresrvation by influencing the activity of regulatory T cells.

Due to well known health benefits of PUFAs, various global organizations and agencies have issued guidelines recommending omega-3 intake and fish consumption to support health and prevent chronic diseases. Despite the importance of omega-3 intake, studies have shown that typical western diets lack sufficient omega-3 fatty acids, with the deficiency being even more severe among populations that are poor or malnourished.

A solution to address omega-3 fatty acid deficiency is through dietary supplementation such as capsules. Supplements are particularly beneficial for individuals who are unable to meet their nutritional needs through diet alone. The use of various forms of omega-3 fatty acids pharmaceutical, nutritional, and dietary products is well established. One example is a concentrated form of long-chain omega-3 PUFAs derived from fish oil, containing DHA and EPA marketed under the trademark Lovaza®. This form is described in U.S. patent numbers 5502077, 5656667 and 5698594.

US7652068 discloses highly purified formulations of omega-3 fatty acids comprising more than 85% omega-3 fatty acids by weight.

US8491953N describes designing a stable food supplement containing fish oil.
US9675575, US10034849 and US10172819 discloses composition, dosage forms and methods of use of DHA and EPA.

Natural bioactives from plants:

A substantial number of plant-derived compounds and traditional remedies have been examined for their lipid-lowering effects, with over 70 medicinal plants demonstrating notable hypolipidemic activity. In recent years, particularly over the past decade, the use of these medicinal plants has grown in urban centers of developed countries. Medicinal plants are recognized as key contributors to hypolipidemic therapy, with reported benefits including high efficacy, a strong safety profile, affordability, and widespread acceptance among users.

Extracts from plants such as Amla (Phyllanthus emblica), Guggul (Commiphora wightii), Black cumin (Nigella sativa), Garcinia (Garcinia cambogia), Green tea (Camellia sinensis) and Frankincense (Boswellia serrata) are recognized for their diverse pharmacological properties, including antidiabetic, hypolipidemic, antibacterial, antioxidant, antiulcerogenic, hepatoprotective, gastroprotective, and chemopreventive effects.

Considering the range of protective benefits fish oil provides in lowering triglyceride levels and protect cardiovascular health and it is appropriate to suggest that a highly bioavailable fish oil formulation combined with other natural compounds exhibiting similar physiological effects, could potentially synergize the cardioprotective effect. This synergistic action could potentially amplify the positive impacts on heart health and lipid metabolism, providing a comprehensive approach to cardiovascular wellness.

Efforts were made to incorporate these plant extracts into our Omega-3 fatty acid formulation to enhance its antihyperlipidemic effect through potential synergistic and/or additive.

interactions.Formulations containing omega-3 fatty acids and plant extracts should exhibit high physical stability and possess optimal physical characteristics to ensure strong patient compliance.

SUMMARY AND OBJECTIVES OF THE INVENTION

This invention pertains to an orally administrable composition comprising omega-3 fatty acids, a plant extract, and at least one surfactant, specifically designed to enhance bioavailability.

Another aspect of the present invention relates to a composition comprising omega-3 fatty acids and one or more plant extracts, wherein the plant extracts are capable of exerting a synergistic and/or additive effect on the anti-hyperlipidemic activity of the omega-3 fatty acids.

Another aspect of the present invention provides a composition suitable for the prophylactic or therapeutic applications commonly associated with omega-3 fatty acids in a subject in need thereof, the composition comprising omega-3 fatty acids, at least one plant extract, and at least one surfactant, formulated for oral administration.

BRIEF DESCRIPTION OF THE DRAWINGS

A comprehensive understanding of some of the features of present invention may be carried out by reference to the following drawings, which display the aspects of invention as observed in preferred embodiments. It should be understood that these drawings are purposed for illustration only and not to limit the scope of invention. The invention is also capable for other embodiments within the scope of appended claims.

FIG 1 is an illustration of the particle size of highly bioavailable omega-3 formulation.
FIG 2 is an illustration of the fatty acid profiling of the highly bioavailable omega-3 formulation.
FIG 3 is an illustration of comparative oral pharmacokinetic profile of the omega-3 fatty acids present in unformulated fish oil with omega-3 fatty acid given as highly bioavailable formulation.
FIG 4 is an illustration of comparative oral pharmacokinetic profile of EPA concentration in Fast state and Fed state of highly bioavailable omega-3 formulation
FIG 5 is an illustration of comparative oral pharmacokinetic profile of EPA concentration in fast state with highly bioavailable omega-3 formulation vs lovaza

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "omega-3 fatty acids" encompasses both naturally occurring and synthetically produced omega-3 fatty acids, as well as pharmaceutically acceptable forms thereof, including but not limited to esters, free acids, triglycerides, derivatives, conjugates, precursors, salts, and combinations thereof. Non-limiting examples of omega-3 fatty acid oils include omega-3 polyunsaturated long-chain fatty acids such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), alpha-linolenic acid (ALA), heneicosapentaenoic acid (HPA), docosapentaenoic acid (DPA), eicosatetraenoic acid (ETA), eicosatrienoic acid (ETE), and octadecatetraenoic acid (also referred to as stearidonic acid, STA). Suitable forms further include esters of omega-3 fatty acids with glycerol (e.g., mono-, di-, and triglycerides) and esters formed with primary, secondary, or tertiary alcohols, such as fatty acid methyl esters and fatty acid ethyl esters. The omega-3 fatty acids, esters, triglycerides, derivatives, conjugates, precursors, salts, and/or mixtures described herein may be utilized in their isolated or pure form, or as components of oils, including but not limited to marine oils (e.g., fish oil, purified fish oil concentrates), algal oils, microbial oils, and plant-derived oils.

As used herein, the terms "fatty acid oil," "omega-3 fatty acid," and "fish oil" may be used interchangeably, except where the context clearly indicates otherwise.

As used herein, the terms "plant extract,""herbal extract", and “bioactive ingredient” may be used interchangeably, except where the context clearly indicates otherwise.

As used herein, "plant extract" refers to an extract, juice, or concentrate derived from any part of a plant, including but not limited to the seed, leaf, fruit, flower, stem, root, tuber, or bark.

"Bioavailability" refers to the measurement of the rate and extent to which a drug or active ingredient reaches the systemic circulation.

As used herein, the term "bioavailability" refers to the "relative bioavailability" between the composition of the present invention, which includes omega-3 fatty acids and plant extracts, and an unformulated omega-3 fatty acid.

COMPONENTS

In one embodiment, a composition is provided that includes omega-3 fatty acids, a herbal extract, an essential oil, an antioxidant and at least one surfactant suitable for oral administration.

In one embodiment, the omega-3 fatty acids may be sourced from marine species, such as fish oil or crustaceans. Examples of fish rich in omega-3 fatty acids include "oily fish" such as salmon, tuna, swordfish, halibut, tilefish, cod (including cod liver oil), and sardines. Additionally, several crustaceans are known to be high in omega-3 fatty acids, including krill (krill oil), and the New Zealand green-lipped mussel, but not limited to them.

In one embodiment, the composition comprises from about 10% to about 70% (w/w) of omega-3 fatty acids relative to the total composition. Preferably, the composition contains about 30% (w/w) omega-3 fatty acids, and more preferably about 60% (w/w) omega-3 fatty acids.

In one embodiment, the omega-3 fatty acid used herein is preferably eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or a combination thereof.

In one embodiment, omega-3 fatty acids, including “EPA” and “DHA”, used herein may be of esterified, triglyceride, phospholipid or free fatty acid forms.

In one embodiment, the omega-3 fatty acids comprising EPA and DHA used herein are preferably in an esterified form.

In one embodiment, the combined content of EPA and DHA constitutes at least 10% w/w, 20% w/w, 30% w/w, 40% w/w,50% w/w, 60% w/w, 70% w/w or preferably about 50% w/w of the total omega-3 fatty acids in the composition.

In one embodiment, the content of EPA alone is at least 22% w/w of the total composition. Preferably, it is above 20% w/w, and more preferably above 25% w/w of the total composition.

In one embodiment, the content of DHA alone is at least 15% w/w of the omega-3 fatty acids in the total composition. Preferably, it is above 17% w/w, and more preferably above 20% w/w of the total composition.

In one embodiment, the composition comprises a herbal extract selected from the group consisting of black cumin extract, resveratrol, garcinia extract, green tea extract, and boswellia serrata extract.

In one embodiment, the herbal extract may exhibit an additive and/or synergistic effect on the antihyperlipidemic activity of omega-fatty acids.

In one embodiment, the herbal extract comprises from about 1% to about 25% (w/w) of the total composition.

In one embodiment, the composition comprises omega-3 fatty acids, a herbal extract, an antioxidant and at least one surfactant.

SURFACTANTS:

A surfactant may, for example, reduce the surface tension of a liquid or the interfacial tension between two liquids. In the context of the present disclosure, surfactants may be used to lower the surface tension between the fatty acid oil mixture and an aqueous solution.

Chemically, surfactants are molecules that possess at least one hydrophilic (water-attracting) region and at least one hydrophobic (lipophilic or oil-attracting) region. Their behavior can be characterized by the hydrophilic-lipophilic balance (HLB) value, which quantifies the relative contributions of hydrophilic and lipophilic properties. The HLB scale typically ranges from 0 to 20, where a value closer to 0 indicates a predominantly hydrophilic character, while a value closer to 20 indicates a predominantly lipophilic character.

In certain embodiments, the surfactant is selected from the group consisting of nonionic surfactants, cationic surfactants, anionic surfactants, zwitterionic surfactants, or combinations thereof.

In certain embodiments, surfactants or emulsifiers are employed in the formulation to enhance the stability, solubility, and bioavailability of the active components. Suitable surfactants include, but are not limited to, polysorbates such as polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). Additional suitable surfactants include sorbitan fatty acid esters (Spans), poloxamers, and phospholipids such as egg lecithin, soy lecithin, and phosphatidylcholine.

Further examples of nonionic surfactants include polyoxyethylene glycol sorbitan alkyl esters, polyoxyethylene stearates, polyoxyethylene castor oil derivatives, polyoxylglycerides, sucrose fatty acid esters, block copolymers of polyethylene glycol and polypropylene glycol, fatty acid esters of ethylene glycol, propylene glycol derivatives (e.g., propylene glycol monolaurate), polyethylene glycol, polypropylene glycol, glycol, trimethylolpropane, and pentaerythritol. Additional surfactant classes may include glucoside derivatives, glycerin alkyl ether fatty acid esters, trimethylolpropane oxyethylene alkyl ethers, fatty acid amides, alkylolamides, alkylamine oxides, lanolin and its derivatives, castor oil and hardened castor oil derivatives, sterols and their derivatives, polyoxyethylene alkyl ethers, polyoxyethylene alkyl allyl ethers, polyoxyethylene alkylamines, polyoxyethylene fatty acid amides, polyoxyethylene alkylolamides, polyoxyethylene diethanolamine fatty acid esters, polyoxyethylene trimethylolpropane fatty acid esters, polyoxyethylene alkyl ether fatty acid esters, polyoxyethylene-polyoxypropylene glycols, polyoxyethylene-polyoxypropylene alkyl ethers, polyoxyethylene-polyoxypropylene polyhydric alcohol ethers, glycerin fatty acid esters, polyglycerin fatty acid esters, polyoxyethylene glycerin fatty acid esters, and Carbomer.

In preferred embodiments, at least one surfactant is selected from the group consisting of polyoxyethylene glycol sorbitan alkyl esters, such as polysorbate 20 (Tween 20), polysorbate 60 (Tween 60), and polysorbate 80 (Tween 80).

In one embodiment, the total amount of surfactant(s) present in the composition does not exceed about 30% w/w of the total composition.

In one embodiment, the formulation may further comprise an antioxidant to enhance the stability of omega-3 fatty acids. Suitable antioxidants include, but are not limited to, ascorbic acid (vitamin C), tocopherols (e.g., a-tocopherol or vitamin E), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate, citric acid, sodium ascorbate, ascorbyl palmitate, rosemary extract, and tert-butylhydroquinone (TBHQ).

In one embodiment, added an essential oil for flavor masking and HDL modulation include, but are not limited to, lemon oil, orange oil, turmeric oil,ginger oil, bergamot oil, basil oil,lemon grass oil and clove oil.

In one embodiments, the composition comprising omega-3 fatty acids, at least one herbal extract, and at least one surfactant may optionally further include one or more co-surfactants, and/or at least one antioxidant.

PARTICLE SIZE:

In one embodiment, the composition may be in the form of a preconcentrate or an emulsion, which facilitates dispersion in an aqueous solution.

In another embodiment composition the composition forms micelles in aqueous medium having, hydrodynamic diameter less than 700nm more preferably hydrodynamic diameter less than 300 nm.

In one embodiment composition comprising omega-3 fatty acids and a herbal extract and an antioxidant and atleast one surfactant for oral administration, wherein the composition forms particle size more preferably hydrodynamic diameter less than 300 nm in aqueous medium.

MANUFACTURING PROCESS:

In another embodiment, the composition pertains to a process for preparing a formulation comprising omega-3 fatty acids, a herbal extract, an antioxidant and at least one surfactant.

The method of preparation involves mixing the omega-3 fatty acids with a surfactant, followed by the addition of herbal extract and the antioxidant, such as vitamin E to the mixture with thorough mixing.

In one embodiment, the pharmaceutical composition of the present invention is encapsulated, preferably in gelatin capsules, which may be either soft or hard. Soft gelatin capsules are typically manufactured and filled in a single, continuous operation.

USE:
Another aspect of the invention provides a composition suitable for the therapeutic treatment for conditions known to benefit from omega-3 fatty acids, intended for a subject in need thereof, wherein the composition comprises omega-3 fatty acids, a herbal extract, an antioxidant and at least one surfactant, and is formulated for oral administration.

Another aspect of the invention provides an omega-3 fatty acid composition comprising herbal extracts, wherein the herbal extracts may exert a synergistic and/or additive effect on the antihyperlipidemic properties of the omega-3 fatty acids.

In another embodiment, the synergistic composition provides both herbal extracts and omega-3 fatty acids together in a stable formulation.

The embodiment provides a composition in which the herbal extracts and antioxidant serve to protect the highly labile omega-3 fatty acid molecules from oxidation, as well as from the detrimental effects of light and air.

In the embodiment, the omega-3 fatty acid in the composition is formulated to be highly palatable, effectively minimizing or eliminating unpleasant odor, aftertaste, and/or burping in the patient.

BIOAVAILABILITY:
The absorption of omega-3 fatty acids in the gastrointestinal tract relies heavily on their emulsification and breakdown into smaller micellar droplets. Bile salts play a crucial role in dispersing large fat globules into these finer droplets. Once emulsified, pancreatic lipase acts on the triglycerides within the droplets, breaking them down into free fatty acids. These smaller, more soluble molecules can then easily pass through the phospholipid bilayer of intestinal cells, facilitating efficient absorption.

The present invention pertains to a self-emulsifying nanoemulsion composition comprising omega-3 fatty acids, at least one herbal extract, and one or more pharmaceutically acceptable surfactants and/or co-surfactants. Upon contact with an aqueous phase under mild agitation—such as that provided by gastric and intestinal motility—the composition spontaneously forms a fine oil-in-water nanoemulsion with droplet sizes typically in the nanometer range (e.g., <200 nm).

This nanoemulsification facilitates the presentation of lipophilic bioactives in a pre-dissolved form, significantly enhancing their solubility, dispersion, and absorption. The resulting nano-sized droplets provide a substantially increased surface area, improving the rate and extent of drug release across the gastrointestinal membrane.

Mechanistically, the enhanced bioavailability conferred by the nanoemulsion system is attributed to:
(i) improved transcellular transport through increased membrane fluidity
(ii) modulation of tight junctions to permit paracellular diffusion
(iii) inhibition of P-glycoprotein (P-gp) and cytochrome P450 (CYP450) enzymes by selected surfactants, thereby reducing efflux and first-pass metabolism
(iv) promotion of chylomicron and lipoprotein formation by the lipid component, enhancing lymphatic transport and systemic delivery of the active constituents.

In one embodiment of the invention, upon oral administration of the pharmaceutical formulation to a mammal, the formulation demonstrates a pharmacokinetic absorption profile—characterized by parameters such as maximum plasma concentration (C_max), area under the plasma concentration-time curve (AUC), and optionally time to reach maximum concentration (T_max)—under fed conditions that is comparable to, or bioequivalent with, the pharmacokinetic profile observed under fasting conditions. In certain embodiments, the mammal is a human.

Accordingly, in evaluating the absorption of omega-3 fatty acids comprising eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)—for example, from omega-3-acid ethyl esters containing EPA ethyl ester and DHA ethyl ester—the increase in the concentration of EPA and/or DHA in plasma or in serum phospholipids may serve as a reliable indicator of absorption. These pharmacokinetic endpoints may further be employed as surrogate measures of oral bioavailability in clinical or preclinical studies.

In other embodiment, the invention provides a method for enhancing the bioavailability of an omega-3 fatty acid in a subject in need thereof, the method comprising orally administering to the subject nanoemulsion composition as described herein, wherein the oral absorption and/or oral bioavailability of the omega-3 fatty acid is improved relative to that of an unformulated omega-3 fatty acid administered under comparable conditions.

In some embodiments, enhanced absorption is evidenced by an increase in the area under the plasma concentration-time curve (AUC) of total omega-3 fatty acids, as measured following oral administration of the composition.

Example 1:
The present invention is further illustrated by representative compositions comprising omega-3 fatty acids in varying ratios of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in combination with one or more surfactants, natural bioactive from plant extracts, and pharmaceutically acceptable excipients. Non-limiting examples of such formulations are set forth in Tables 1. These compositions may be prepared by standard nanoemulsion formulation techniques, including but not limited to, homogenization, low- or high-energy emulsification, solvent evaporation, ultrasonication or spontaneous emulsification methods. In a typical process, the omega-3 fatty acid source is blended with surfactants, co-surfactants, an antioxidant, an essential oil and plant extracts under gentle mixing to yield a formulation, which upon dilution with an aqueous medium, forms a fine emulsion or nanoemulsion suitable for oral administration.

The invention is further illustrated by the following examples, which are not intended to limit the scope or composition of the present invention.

Example 1:
The following examples illustrate compositions containing omega-3 fatty acids with varying amounts of EPA and DHA, along with surfactants, natural bioactives from plants, and other excipients, as detailed in Tables 1–7.
Sl.No Ingredient Percentage
Table 1
1 Fish Oil 20-60%
2 Amla extract 10-40%
3 Vitamin E 0.5-2%
4 Polysorbate 80 20-40%
5 Essential oil 0.5-2%
Table 2
1 Fish Oil 20-60%
2 Resveratrol 10-40%
3 Vitamin E 0.5-2%
4 Polysorbate 80 20-40%
5 Essential oil 0.5-2%
Table 3
1 Fish Oil 20-60%
2 Garcinia extract 10-40%
3 Vitamin E 0.5-2%
4 Polysorbate 80 20-40%
5 Essential oil 0.5-2%
Table 4
1 Fish Oil 20-60%
2 Green tea extract 10-40%
3 Vitamin E 0.5-2%
4 Polysorbate 80 20-40%
5 Essential oil 0.5-2%
Table 5
1 Fish Oil 20-60%
2 Thymoquinone 10-40%
3 Vitamin E 0.5-2%
4 Polysorbate 80 20-40%
5 Essential oil 0.5-2%
Table 6
1 Fish Oil 20-60%
2 Guggul Extract 10-40%
3 Vitamin E 0.5-2%
4 Polysorbate 80 20-40%
5 Essential oil 0.5-2%
Table 7
1 Fish Oil 20-60%
2 Boswellia Serrata Extract 10-40%
3 Vitamin E 0.5-2%
4 Polysorbate 80 20-40%
5 Essential oil 0.5-2%

ANTIOXIDANT ACTIVITY STUDIES

Example 2
Illustrates antioxidant potential and free radical scavenging activity of omega 3 formulation was detected by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay. DPPH radical was reduced to diphenylpicrylhydrazine upon interaction with omega 3 formulation. Decolourization was measured at 517nm and percentage of radical scavenging activity was calculated. Comparative analysis of IC50 values are calculated for standard – ascorbic acid and omega 3 formulation in correspondence to their specific graphs.

Table 8-In vitro biochemical antioxidant assay (DPPH- 1,1-diphenyl-2-picrylhydrazyl) IC50 values of Ascorbic acid and Omega-3 formulation.
Sl No. Sample name m-slope c-intercept IC50
x = (y-c)/m,
y=50
1 Ascorbic acid 5.518 39.944 1.82
2 Omega-3 formulation 6.1942 5.505 4.81

Example 3
Illustrates antioxidant activity of omega 3 formulation by FRAP assay

The antioxidant capacity of the omega 3 formulation was assesses using Ferric Reducing Antioxidant Power (FRAP) Assay. Omega 3 formulation reduced ferric ions (Fe3+) to ferrous ions (Fe2+) when mixed with FRAP reagent – mix of ferric chloride, 2,4,6-tripyridyl-s-triazine and acetate buffer. Blue colour upon reduction was measured at 593 nm. The antioxidant capacity was expressed in ferric ion reducing ability or Fe2+ equivalence from standard graph of ferrous sulphate. The ferric ion-reducing ability of the omega 3 formulation is equivalent to 146.5 µM of ferrous ions.

RELATIVE BIOAVAILABILITY STUDIES

Example 4
Enhanced bioavailability (FIG 3)
A comparative pharmacokinetic study of the omega-3 formulation in the invention against fish oil with similar omega-3 fatty acid content was carried out in Sprague dawley rats at unimolar dose i.e similar amount of omega-3 fatty acid was administered to the different groups treated with omega-3 formulation and the unformulated fish oil. The design of study offered a weight-to-weight comparison of the omega-3 fatty acid bioavailability given as novel formulated omega-3 and unformulated fatty acid. The collection of the blood carried out at defined time interval and plasma separated from it by centrifuging at 4000 rpm speed for 10 min. The plasma samples were analyzed using a validated LC-MS/MS method.

Table 9 -Results in Table 9 shows improvement in Cmax for the omega 3 formulation prepared according to our invention.
Comparative bioavailability results of formulated and unformulated Omega-3 fatty acids
Formulated Unformulated
Cmax Tmax Cmax Tmax
11.3281 1.89 16.0663 3.513
AUC0-t 41.039 AUC0-t 16.066

Example 5
Illustrates relative bioavailability studies of omega – 3 formulation as 0.5g soft gelatin capsules prepared accordingly under fast and fed conditions (FIG 4).

Table 10 - The results presented in Table 10 indicate that the oral pharmaceutical composition containing omega-3 fatty acids exhibits an increased area under the curve (AUC?) under fed conditions as compared to fasted conditions.
Omega – 3 formulation 0.5g capsules
(under fasting conditions) Omega – 3 formulation 0.5g capsules
(under fed conditions)
Cmax Tmax Cmax Tmax
1667.51 4.2 1413.98 5
AUC0-t 6972.21 AUC0-t 8074.52

Example 6
Relative bioavailability in fasting condition

Relative bioavailability studies of omega-3 formulation, 0.5g prepared according to given examples and lovaza® (omega-3-acid ethyl esters) capsules 1 g of Glaxosmithkline, RTP, NC 27709 (four capsules as single dose) in health, adult, human subjects under fasting condition is studied. The statistical analysis was not performed as there are no EPA concentrations obtained for lovaza under fasting conditions. EPA concentration of lovaza® vs omega-3 formulation in fasting condition is illustrated in FIG: 5. ,CLAIMS:1. A nutraceutical composition comprising:

a) Omega-3 polyunsaturated fatty acids (PUFAs) comprising eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in a ratio of 28:20;
b) A natural bioactive compound derived from a plant oil, constituting 15–25% of the oil component; and
c) A nanoemulsion delivery system with a mean particle size of 200–300 nm and zeta potential =-30 mV,
wherein said composition exhibits synergistic antioxidant and anti-inflammatory activity for cardiovascular support.

2. A method of treating cardiovascular disease comprising administering a therapeutically effective amount of a nanoemulsion formulation containing:

i) 28–30% EPA and 20–22% DHA from fish oil;
ii) 15–25% natural bioactive compound derived from a plant extract and
iii) A (Generally Recognised as Safe) GRAS emulsifier stabilizing droplets with hydrodynamic diameter <300 nm,
wherein the formulation reduces serum triglycerides by =25% and LDL cholesterol by =15% within 4 weeks.

3. The composition of claim 1, further comprising 0.1–2% vitamin E as a stabilizing antioxidant and additionally contributes to the reduction of serum triglyceride levels upon oral administration.

4. The composition of claim 1, wherein the emulsifier is polysorbate 80 at 5–25% w/w.

5. The composition of claim 1, having a DPPH radical scavenging IC505of =5 µg/mL and FRAP value =100 µM Fe²? equivalence.

6. The composition of claim 1, formulated for oral administration with bioavailability (AUC) =2-fold higher than unemulsified EPA/DHA in fasted states.

7. The composition of claim 1, wherein natural bioactive compound from plant extract potentiates PPAR-? activation by EPA/DHA, reducing NF-?B-mediated inflammation.

8. he method of claim 2, wherein the formulation achieves plasma EPA concentrations =150 ng/mL within 2 hrs post-administration under fasting conditions.

9. The composition of claim 1, further comprising essential oil at 0.5–3% w/w for flavor masking and HDL modulation.

10. The composition of claim 1, exhibiting =3% moisture content and viscosity 40–60 cP for shelf stability =24 months.

11. The composition of claim 1, packaged in enteric-coated softgels for targeted intestinal release.

12. The composition of claim 1, wherein the nanoemulsion enhances cellular uptake in endothelial cells by =50% compared to bulk oil.