FIELD OF THE INVENTION
The invention relates to a formulation for a nutraceutical product and its process of preparation thereof. The invention in particular relates to a nutraceutical nanoformulation product and a process of its preparation by utilizing agrowaste specifically kinnow peel waste.
BACKGROUND OF THE INVENTION
Kinnow mandarin, a hybrid of Citrus nobilis and Citrus deliciosa, was developed by H.B. Frost at California, USA in 1935. It has been grown commercially ever-since in California, Arizona, West Pakistan, and India (Punjab). It has become a major citrus fruit due to its high juice content and better quality as compared to other citrus fruits.
Kinnow is also known to be full of phytochemicals like terpenoids (limonoids and carotenoids) and polyphenols (flavonoids and phenolic acids). These compounds exhibit several anti-allergic, anti-inflammatory, anti-cancer, anti-viral and antibiotic (antimicrobial) properties. Other than these Kinnow mandarin is a good source of Folic acid, ß-carotene etc. Folic acid is recommended for all women of childbearing age to prevent serious birth defects. While ß-carotene (which is source for vitamin A) is important in immune response and acts as antioxidant.
This citrus crop is mainly utilized in the fruit industry as a source of healthy juice, but the residual leftovers after juice production possess several clean-up issues and eventually cause environmental stress. Therefore, in contrary to the current practice, the method of the present invention utilizes these leftover citrus peels for human and animal benefaction. These citrus peels and seeds contain many phenolic compounds and according to a study by Salmah Yusof et al (1990) citrus peels are richer in flavonoids than the seeds.
Therefore, the process of the present invention overcomes the drawbacks of the prior art and offers an improved and economical way of waste utilization with lots of health benefits as well.
SUMMARY OF THE INVENTION
The present invention provides for a stable nanoformulation of kinnow peels by processing the left-over wastes. The formulation according to the present invention enhances the bio-availability of the nutraceutical components (limonoids, flavonoids and carotenoids) extracted from the kinnow peels by converting them into nanoformulations. These nanoformulations help in maintaining the bio-activity of these bio-molecules as they remain intact without undergoing any premature metabolism in the gastro-intestinal tract.
The present invention also provides a nanoformulation of lipophilic agro waste extract with enhanced bioavailability comprising in percentage by weight; 2-20% of lipophilic agro waste extract wherein the lipophilic agro waste extract is derived from kinnow peels by SCF-CO2 extraction process; an Organic phase having fatty acid or fatty acid derivatives in the range of 0.5-30% and optionally comprising edible oil : 0-5%; and an aqueous dispersion phase having surfactant: 2-3%, co-surfactant: 40-60%, and remaining as water; wherein the nanoformulation has hepatoprotective and antioxidative properties.
The present invention also provides nanoformulations containing the kinnow peel extract for use in poultry industry, where the formulation on administration to the birds, gets absorbed into the blood stream and the phytochemicals get translocated to different parts of the body, leading to two significant outcomes: (i) increase in muscle mass (ii) removal of all antibiotic from poultry feed.
The present invention also provides a process of preparing a nanoformulation comprising lipophilic agro waste kinnow peels extract, the process consisting of: Subjecting the kinnow peels to supercritical fluid carbon dioxide (SCF-CO2) to extract a solution rich in limonoid concentration; Subjecting the kinnow peels to supercritical fluid carbon dioxide (SCF-CO2) to extract another solution rich in carotenoids and flavonoids concentration; mixing the SCF-CO2 extracted kinnow peel extracts obtained in step a) and step b) to yield a solution having the required amounts of limonoids, carotenoids and flavonoids concentration wherein the amount of kinnow peels extract ranges from 0.5 to 30% (in percentage by weight) of the final formulation. Stirring the solution obtained in step c) at 40oC to ensure uniform mixing and optionally adding edible oil: 0-5% in percentage by weight of the final formulation; preheating the fatty acid or fatty acid derivatives (0.5-30% in percentage by weight of the final formulation) to its melting point and adding the required quantity of solution obtained in step d) to it, stirring and heating the mixture obtained in step e) at 75 °C on magnetic stirrer to form a solubilized and uniform organic phase solution; Adding an aqueous phase comprising surfactant: 2-3%, co-surfactant: 40-60%, and the balance of water (in percentage by weight of the final formulation) dropwise to the solution obtained in step f) under stirring conditions at 75 °C. Subjecting the mixture of step (b) to Ultrasonic homogenization to obtain complete solubilization of the organic phase in the aqueous phase. Wherein the Ultrasonic homogenization is carried out with 6mm probe at 25-35% amplitude, pulse 25s on/5s off for 20 minutes at 75 °C.
The present invention also provides for an economical process of agro-waste utilization that leads to the production of an animal and human health wellness product.
The present invention also provides a process of utilization of natural biomass to develop a product capable of treating fatty liver and alcoholic hepatitis. These liver conditions, over long periods of time tend to develop into liver cancer.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1. Illustrates a histopathology of the liver of poultry birds with different doses of SLNs (solid lipid nanoparticles) and KPP (kinnow peel power).
Figure 2. Illustrates a Lipid Blend Characterization and Process Parameter Optimization for Bioengineering the Nutraceutical Product
DETAILED DESCRIPTION OF THE INVENTION
In the following description, various aspects of the invention will be described. For the purposes of explanation, specific details are set forth in order to provide a thorough understanding of the invention. Alterations and further modifications of the inventive features, and additional applications of the principles of the invention as have been illustrated herein, which would occur to one, skilled in the relevant art and having possession of this disclosure, are to be considered within the scope of the invention.
The invention adds to human knowledge in the realm of health promoting foods (nutraceuticals) derived from natural biomass. The invention provides for an economical process of agro-waste utilization of the residual leftovers after kinnow juice production which possess several clean-up issues and eventually environmental stress.
Citrus agrowastes mainly comprise two types of terpenoids: limonoids and carotenoids. Of these, limonoids occur significantly in the juice as glyconic forms in the peels and seeds in the agyconic (bitter) form.
Many studies show that kinnow peels are rich in phenolic compounds, which are well known as polyphenols. These polyphenols have antioxidant properties due to their radical scavenging activity or they act as free radical terminators. The antioxidants play a major role in human body but for a healthy body, there must be a balance between antioxidants and pro-oxidants. Diversion of the ratio towards pro-oxidants leads to oxidative stress in the body. This oxidative stress can give rise to severe disorders like malignancies, cardiovascular disorders, diabetes, neurological disorders, renal diseases, aging, liver or respiratory disorders, skin diseases, inflammatory disorders etc. The formulation of the present invention provides for an anti-oxidant rich health supplement.
The present invention further provides for a stable nanoformulation of kinnow peels extract rich in nutraceutical components namely limonoids, flavonoids, carotenoids or the like.
Limonoids are a group of secondary metabolites of citrus fruits. “Limonoid” term was derived from limonin, which is a tetranortriterpenes. The biological activities of these compounds have indicated an improvement in human heath as it has insecticidal, antibiotic (antimicrobial), anti-malarial, anti-viral, anti-cancerous and hepatoprotective activities and other pharmacological activities. The two main limonoids identified in kinnow mandarins are limonin and nomilin.
Flavonoids are highly diversified plant pigments, present in vegetables, fruits and several other plant parts. Flavonoids are known to have anti-oxidative activity, coronary heart disease prevention and anti-cancerous activity and free radical scavenging and immune modulation capacity. The main flavonoids identified in kinnow mandarins are tangeritin and nobiletin, besides phenolic acids, chlorgenic acid, ferulic acid and caffeic acids which impart significant anti-oxidative capacity.
Carotenoids are a group of fat-soluble pigments present in many fruits and vegetables. These are broadly classified into: carotenes and xanthophylls. Carotenoids provide many health benefits for example preventing risk of certain cancers and eye diseases, since carotenoids acts as anti-oxidants and beta carotene being a provitamin A (i.e) can be converted into vitamin A. Xanthophylls (Lutein and zeaxanthin) also act as antioxidants and have the ability to absorb the blue light which can be damaging to the eye.
All these three classes of nutraceuticals, namely limonoids, flavonoids and carotenoids have poor bioavailability, which is attributed to their structure; passive diffusion and active efflux by the intestinal cells because of the lack of specific receptors.
The present invention further specifically provides bioavailable forms of these nutraceuticals in the body that can provide various health benefits. The nanoformulations according to the present invention help in improving the bio-availability and thus enhancing the bioactivity of these compounds.
In food industries, it has become clear that during the development of health promoting foods, inclusion of these compounds into the food matrices is a major problem, as these compounds are highly lipophilic in nature which leads to strong binding of these bioactive compounds to the matrices which in-turn results in poor absorption and bio-availability in the body.
Therefore, the methods and processes of the present invention overcome these limitations with a wide variety of delivery systems such as solid lipid nanoparticles, lipid blend based nanoparticles, nanoemulsions, etc. These delivery systems involve varying content proportions in solid/liquid lipid carriers. These systems have improved the efficacy of the encapsulated compounds. The formulations according to the invention may comprise a mixture of the active compound with surfactants, oil and/or a single or mixture of sustained release polymers; or the active compound with a single or mixture of sustained release polymers and a lipid component to enhance the release properties of the formulation.
Further according to other embodiments of the present invention the formulations of the invention can be combined with other carriers to achieve the desired outcome. These formulations may be delivered to or can be adapted for, oral, nasal, buccal, ocular or topical delivery. However, oral delivery is mostly used. The formulations can be consumed alone or may be supplemented with other pharmaceutical or nutraceutical compounds to achieve the desired results.
According to an exemplary embodiment, the present invention provides a nanoformulation of lipophilic agro waste extract comprising: 2-20% lipophilic kinnow peel waste extract; 0.5-30% an organic phase having fatty acid or fatty acid derivatives and/or 0-5% edible oil; and an aqueous dispersion phase having 2-3% surfactant, 40-60% co-surfactant, and the balance of water. The lipophilic kinnow peel waste extract is derived by SCF-CO2 extraction process. The fatty acid used in the formulation is selected from, but not limited to, Stearic acid, stearic acid methyl ester and stearic acid (stearic acid ethyl ester) ethyl ester (stearic acid methyl ester), Dodecanoic acid, Myristic acid, Palmitic acid and their combination thereof. Similarly, the surfactant and co-surfactants used in the formulation is selected from the group of non-ionic surfactants, but not limited to, Tween 20, Tween 80, Span 20, Span 80, Span 85, Tyloxapol, Poloxamer 188, Poloxamer 407, Poloxamine 908, Brij78, Tego care 450, Solutol HS15, Polysorbates, ethanol, 1,2-propanediol, Glycerol, and other food/ pharma grade surfactants.
The present invention further relates to a process of making a nutraceutical nanoformulation comprising bioengineering of the product by high energy emulsification; using solid and liquid lipids as nanocarriers and encapsulation of the Supercritical fluid CO2 extracts (SCF-CO2 extracts) of kinnow peels to form a nanoformulation. Also, the balance between enhancing the bio-dispersibility of the product with respect to the size dimensions of the formulations has been monitored during the process. This ensures that the nanodroplet enters the intestinal cells, as well as refrains from entering the blood brain barrier. SCF-CO2 extraction system helps retain the valuable bio-active components in the kinnow peels such as flavonoids, limonoids, carotenoids or the like.
SCF-CO2 extract preparation:
SCF-CO2 extraction system extracts chemical compounds using supercritical carbon-dioxide instead of organic solvents. Supercritical extraction has been particularly useful in extracting lipids and aromatic components from the plants for example coffee industry relies on these processes by which caffeine can be removed from coffee beans. The oil industries use supercritical processes for removing oils from cotton seeds, soyabean, ginger and corn. Flavor extraction using this process has been applied to fruits, vegetables, flowers and spices. Other than these SCF-extraction is used for deriving by-products, antioxidants, vitamins, plant metabolites from the fruits and vegetable waste.
According to another embodiment of the present invention, the kinnow peel extract is extracted by the supercritical fluid extraction system in two sets. One for the extraction of a solution rich in limonoids (limonins) and the other is for the extraction of a solution rich in carotenoids and flavonoids.
In the supercritical fluidic system, a gas is subjected to a pressure that is sufficient to provide its density equal to liquid density, the pressure exerted at a temperature above the critical temperature of the gas. Critical temperature is the temperature above which pressure liquification cannot occur. These supercritical fluids and their respective operative conditions such as their critical temperatures and pressures are well known in the art. The gases from which supercritical fluid can be derived are selected from the group of, but not limited to, carbon dioxide, methane, ammonia, ethane, water, 2,3-dimethylbutane, benzene, diethyl ether, toluene, n-propane, n-butane, n-pentane, n-hexane, n-heptane, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and chlorotrifluoromethane. Carbon dioxide is the preferred gas for practicing this invention.
According to another exemplary embodiment of the present invention, for the first set, the system is operated for 4 hours, at 350 bar and 55°C for the extraction of limonoids out of kinnow peels. Similarly, for the second set, the system is run for 4 hours at 330 bar and 40°C with additional 8% co-solvent selected from the nonlimiting examples of methanol, propylene glycol, acetone for the extraction of carotenoids and flavonoids. The extracted products from the above two sets of extractions were mixed together to obtain the required concentration of limonoids in the test sample extract.
Protocol for the preparation of nanoformulation product of the kinnow peel extract
The present invention provides the process of preparation of a nanoformulation comprising lipophilic agro waste kinnow peels extract, the process consisting of:
a) Extracting a solution from the kinnow peels, in the above described SCF-CO2 system, where the extracted products from the above two sets of extractions were mixed together to obtain a final kinnow peel extract which is rich in limonoids, carotenoids and flavonoids concentration, wherein the amount in weight percentage of kinnow peels extract ranges from 0.5 to 30% of the final formulation. b) Stirring the solution obtained in the previous step at 40°C to ensure uniform mixing and optionally adding edible oil: 0-5% in percentage by weight of the final formulation. Edible oil in the process is selected from the nonlimiting examples of olive oil, coconut oil, canola oil, fish oil and mixture thereof. c) Next step is to preheat the fatty acid or fatty acid derivatives (0.5-30% in percentage by weight of the final formulation) to its melting point and add the required quantity of solution obtained in step b) to the melted fatty acid or fatty acid derivatives. Nonlimiting examples of the fatty acids are stearic acid, stearic acid methyl ester and stearic acid (stearic acid ethyl ester) ethyl ester (stearic acid methyl ester), Dodecanoic acid, Myristic acid, Palmitic acid and their combination thereof. d) Stirring and heating the mixture obtained in step c) at 75°C on magnetic stirrer to form a solubilized and uniform organic phase solution. e) Adding an aqueous phase comprising surfactant: 2-3%, co-surfactant: 40-60%, and the balance of water (in percentage by weight of the final formulation) dropwise to the solution obtained in step d) under stirring conditions at 75°C. Surfactants and cosurfactants for the process disclosed in the present invention are selected from the group of, but not limited to, Tween 20, Tween 80, Span 20, Span 80, Span 85, Tyloxapol, Poloxamer 188, Poloxamer 407, Poloxamine 908, Brij78, Tego care 450, Solutol HS15, Polysorbates, ethanol, 1,2-propanediol, Glycerol, and other food/ pharma grade surfactants. f) Subjecting the mixture of step (e) to Ultrasonic homogenization to obtain complete solubilization of the organic phase in the aqueous phase.
The nanoformulation according to the present invention can be used as dietary, nutraceutical and health supplement, either alone or in combination with/ or as a part of a diet, food, feed etc.
The present invention also provides nanoformulations containing the kinnow peel extract for the use in poultry industry, wherein the nanoformulation on administration to the birds, gets absorbed into the blood stream and the phytochemicals get translocated to different parts of the body, leading to two significant outcomes: (i) increase in muscle mass (ii) removal of all antibiotic from poultry feed.
The present invention also provides a nutraceutical product possessing hepatoprotective and anti-hepatotoxicity activity, and is capable of treating fatty liver and alcoholic hepatitis. These liver conditions, over long periods of time tend to develop into liver cancer. The limonoids which are the main component of the kinnow peel extract, are known to exert a valuable antioxidant activity along with the carotenoids and flavonoids. This antioxidant activity can contribute to the therapeutical effect for many lifestyle diseases like hypertention, heart diseases, diabetes, stroke and septic shock, cancer, rheumatoid arthritis, myocardial infarction, cardiovascular diseases, chronic inflammation, aging and other degenerative diseases in humans.
According to another embodiment of the invention, the concentrations of the nutraceutical components (namely limonoids, flavonoids, carotenoids or the like) in the nanoformulation plays a critical role for the desired outcome. This requires a dose optimization with respects to these components. In one specific embodiment the dose optimization with respect to limonoids is done. It requires the supercritical fluid extract from two completely different sets of conditions to be mixed to obtain a concentration of about 30-45 mg limonoids per ml of extract, whilst retaining a substantial concentration of flavonoids and carotenoids.
The following examples illustrate the aspects of the present invention:
Example 1:
In one exemplary embodiment, the nanoformulation of the kinnow peels extract, comprising: 4.3% lipophilic agrowaste extract (kinnow peel extract), 58.5 % glycerol, 25.7% stearic acid, 3% Tween-20 and 8.5% of water, can be administered to poultry birds in the form of SLN (solid lipid nanoparticles). The process involves the preparation of the organic phase comprising 0.433 g of SCF-extract and 2.567 g of stearic acid; an aqueous phase (approx 12 g) containing 5.85 g of water, 5.85 g of stearic acid and 0.3 g of Tween- 20. The organic phase is stirred on magnetic stirrer and aqueous phase is added into it under stirring, for 15 minutes, at the temperature of 75 °C. After the mixing, ultrasonication is done with 6 mm probe at pulse of 25s on/5s off for 20 minutes at 35% amplitude (keep temperature 75°C using oil bath). The process is maintained at the temperature of 75 °C.
To study the activity of the nanoformulation derived from SCF-CO2 kinnow peel extract according to the present invention, it was fed to poultry birds and a comparative study was deduced from it. The different SCF-CO2 extracts of kinnow peels were mixed in the specific ratios to obtain a final limonoid concentration of 30mg/ml in the nanoformulation. Accordingly, the dose for poultry birds was kept in three variants, that is 0.5 mg/bird/day, 1.0 mg/bird/day and 1.5 mg/bird/day.
Accordingly, the comparative study design comprised:
Set of poultry birds; fed with
a) Test sample: Limonin Solid Lipid Nanoparticles (SLNs).
b) Negative control: Blank Solid Lipid Nanoparticles (SLNs) - contain all components used in the preparation of SLNs, except the bioactive component under investigation i.e. Limonins.
c) Positive control: Kinnow Peel Powder, dosages- 0.5 mg, 1.0 mg, 1.5 mg per bird per day.
The dose of limonins in positive control and test sample was equal, each containing 7.47mg limonoids per gm of sample.
Histopathology of the bird’s liver was done for all set of birds fed with different doses.
Results:
According to the above study conducted on poultry birds, significant trans-location of limonin to the muscle was identified in 0.5 mg and 1.0 mg dosages of SLNs, and accumulation of limonin in the liver was observed with the higher dose (i.e.) 1.5 mg SLNs, indicating enhanced bioavailability with the nanoformulation. The study suggested that no translocation of limonins was observed in the birds, when fed with the conventional kinnow peel powder.
Table 1: shows the comparative data for the translocation of the limonine in the muscles and the liver of the birds.
Sample Code No. Limonins detected in Meat (M)
(mg/g of tissue) Limonins detected in Liver
(mg/g of tissue)
Group 1 : Negative control: Blank SLNs
4706 ND ND
4605 ND ND
Group 2 : Positive control: 0.5 mg
4646 ND ND
4642 ND ND
Group 3 : Positive control: 1.0 mg
4637 ND 60.4
4631 ND 84.4
Group 4 : Positive control: 1.5 mg
4614 11.93 29.96
4625 18.0 31.06
Group 5 : Test SLNs: 0.5 mg
4773 45.51 20.33
4782 27.13 17.24
Group 6 : Test SLNs: 1.0 mg
4770 36.15 19.08
4768 46.0 22.36
Group 7 : Test SLNs: 1.5 mg
4758 17.07 79.33
4756 32.56 86.44

Meat limonins (M) to be considered as sum of Breast limonins and Leg Limonins, as biochemically analyzed. (ND) indicates that limonin is “not detected” in the sample.
It is clear from the table 1, there is no or little translocation of limonins using kinnow peel powders. The inference is based on translocation of limonins from liver to meats and the effect of physical form of supplementation (kinnow peel powder or solid lipid nanopartcles) on the same, if any.
Furthermore, the histopathology of bird liver, as shown in figure 1; confirmed that when birds were fed with conventional kinnow peel powder (KPP), there was infiltration of mononuclear inflammatory cells in the parenchyma, especially around small blood vessels and congestion in the central vein. Whereas supplementation of diet with nanoformulations (SLN-Solid Lipid Nanoparticles) revealed almost normal histo-architecture, particularly at low dosages.
The observations from the figure 1. indicate that nanoformulations @1.0mg/bird/day is an effective carrier of limonins, leading to improved growth and health characteristics in birds.
Example 2:
In another exemplary embodiment, the nanoformulation of the kinnow peels extract, comprising: 16.67% kinnow peel extarct, 43.33 % glycerol, 0.56% stearic acid, 2.22% Tween-20, 5% olive oil and 8.5% of water can be administered to human beings in the form of NLB (Nano Lipid blend). The process involves the preparation of the organic phase comprising 3 g of SCF-extract, 0.9 g of olive oil and 0.1 g of melted stearic acid. The organic phase is prepared by first heating and mixing the SCF-extract of kinnow peels and olive oil on a magnetic stirrer at 40 °C, followed by addition of the melted stearic acid to the above mixture. The process further comprises an aqueous phase containing 2.5% tween 20 in glycerol: water, 1:1, v/v. The aqueous phase is added dropwise to the above organic phase under stirring conditions for 10 minutes. Immediately, after the mixing, ulltrasonication is done with 6 mm probe at 25% amplitude, pulse 10s on/20s off for 10 minutes at room temperature.
A pilot study conducted on the humans with a set of objectives:
a) Improvement in quality of life
b) Improvement in liver function test.
Human recipients were administered with the following set of doses of nanolipid blend of kinnow peel extract.
a) Positive control: LimA; only kinnow peel powder, not extract.
b) Negative control: LimB; Blank Nanoformulations; - contains all components used in the preparation of the nanolipid blend, except the bioactive component under investigation i.e. Limonins.
c) Test sample: LimoC; Nanoformulation- contains all components used in the preparation of the nanolipid blend, including the limonins.
The dose for the nanoformulation for the human being was optimized with respect to the dose of nanoformulation for poultry birds. The calculations involved average weight of human to average weight of the bird with the dose consumption per day by the poultry birds. Based on these calculations the dose for human beings was fixed to 5.0 mg/ml twice a day. Therefore, the dose of limonoids in positive control and test sample was equal, each containing 5mg limonoids per ml of sample.
Results:
According to the above study indicates a favourable trend and the association is good enough between giving oral nanolipid blend, and improvement in liver functions of patients with non-alcoholic fatty liver.
Table 3: Preliminary results of liver function tests-AST and ALT, done on patients with non-alcoholic fatty liver
S. No.
Patient Id Weight
(Baseline) AST
(Baseline) ALT
(Baseline) Weight
(2 Months) AST
(2 Months) ALT
(2 Months)
1 LIM-A-01 87.2 30 35 86.4 40 34
2 LIM-A-02 98.8 103 118 96.6 94 99
3 LIM-A-03 86.6 75 39 85.4 41 35
4 LIM-B-01 79 29 47 78 28 37
5 LIM-B-02 75 36 41 74 36 38
6 LIM-B-03 119 50 37 110 30 29
7 LIM-C-01 78.3 83 111 77 40 67
8 LIM-C-02 88 48 102 86 40 95
9 LIM-C-03 84.6 42 52 83.7 36 37

Liver toxicity is used to be measured with a blood test, which reveals the ratio of the concentration of the enzyme aspartate transaminase (AST) and alanine transaminase (ALT) in the blood of a human or animal. An AST to ALT concentration ratio of 2:1 or greater is suggestive of alcoholic liver disease.
The above results of the pilot study showed promising results with the LimC-01, LimC-02, LimC-03 for the AST/ALT ratios in the time period of 2 months.
The trend on AST/ALT with Test (C) is encouraging for secondary objective (a) that is improvement in the quality of life. The Pilot Study shows a favourable trend.
The embodiments of the present invention are not limited to the above examples. The above embodiment may include other examples of the nanoformulation, which are formulated keeping in view the FSSAI regulations for human consumption. Exemplary other formulations in accordance with the embodiment of the present invention are listed in Table in figure 2. These and the above are not limited in this regard and, in various implementations, these and the above examples may include composition and preparation of the nanoformulation serving the purpose of a dietary supplement, nutraceutical, health supplement, ayurvedic medicine, over the counter product(s), undertaking different sets of features, different order of such features and/or additional features other than those describes explicitly in the invention.
The nanoformulations according to the embodiments of the present invention exhibit enhanced stability. The nanoformulation of the kinnow peel extract, prepared from the methods and processes described in the present invention showed no turbidity or precipitation and had a good stability of atleast over time of 1 year. The size of nanoparticle ranges from 160 to 250nm for increased aqueous dispersion and enhanced efficacy for bio-availability.
Table 4. Stability of nanoformulation of kinnow peel extract, in-particular the nano-lipid blends for humans according to embodiments of the present invention.
Time duration Particle size PdI ?-potential
1st month 186.07±3.27 nm 0.181±0.05 -67.90±1.73 mV
2nd month 217.27±8.68 nm 0.172±0.035 -62.17±1.55 mV
3rd month 183.67±4.91 nm 0.115±0.034 -82.00±0.35 mV
4th month 181.86±2.63 nm 0.113±0.092 -74.93±2.58 mV
5th month 188.10±3.05 nm 0.081±0.040 -93.73±1.98 mV
6th month 181.27±2.91 nm 0.209±0.037 -66.5±3.03 mV
7th month 180.33±12.7 nm 0.122±0.111 -68.87±0.40 mV
8th month 200.07±24.17 nm 0.427±0.158 -67.50±1.47 mV
9th month 166.85±1.63 nm 0.462±0.217 -61.13±0.71 mV
10th month 181.60±7.07 nm 0.072±0.023 -68.30±2.97 mV
11th month 174.43±11.19 nm 0.093±0.034 -54.37±1.79 mV
The product of the present invention offers immense and long-term commercial value. The present invention provides a nutraceutical formulation derived from kinnow peel extracts. Under the FSSAI- Food Safety and Standards Authority of India, it satisfies all criteria to be classified as a nutraceutical. After the characterization of the extract, it is apparent that it contains substantial concentrations of Limonoids (chiefly Limonin), followed by bioflavonoids and biocarotenoids, a complement which makes it a health promoting combination of natural bioactives. The product from the present invention is named LimoPan, for which the trade mark has been filed and is in the processing stages of approval.
The nanoformulation of the present invention provides a good stability and bioavailabilty over the conventional kinnow peel powder. The nanoformulation can enhance the stability of kinnow peel extract’s components that are limonoids, carotenoids and flavonoids in the preparation process, improve their solubility and bioavailability when administered to humans or animals. Present invention also provides for highly sustained release nanoformulations of kinnow peel extract, capable of delivering significantly higher amount of active molecules that is limonoids to the human and/or animal system, thus rendering enhanced therapeutic efficacy. These nanoformulation has wide market prospects in the medicine field.
Advantages of the invention:
The present invention provides two distinct advantages:
(a) Agrowaste utilization: The process and the method of the present invention aids sustainable agriculture by utilization of the quantum biomass generated from the industry, as agrowaste, a feature of particular importance to agrarian nations. It is imperative for this biomass to be utilized since it is a storehouse of nutraceuticals, and this invention has developed a product from this biomass.
(b) Animal and human wellness: A comparative data on improved health of the poultry birds fed with the nanoformulations vis-à-vis dried kinnow peel powder highlights the enhanced role of the nanoformulation. The data shows the translocation of the nutraceutical (limonin in particular) from liver to the muscles reiterating the fact that the nanoformulations lend enhanced bioavailability. As it can be seen from the study above that this enhanced bioavailabilty led to improved health of the birds, in terms of weight gain and without the need of any antibiotic in the feed. For the purpose of human consumption, keeping in view the FSSAI regulations, this nanoformulation was re-formulated, replacing in part the solid lipid with edible olive oil, maintaining the characteristics of the nanoparticles. The details are listed in Table 2.
(c) For the treatment of other lifestyle diseases e.g. Diabetes, blood pressure, atherosclerosis, heart disease etc. Studies on human have shown that there was a significant decrease in the blood glucose level of the patient with the defined doses of limoPan.
It is to be understood that the process for preparation of Nanoformulations and the product thereof is not limited to the processes described here within, it is to be noted that various changes and modifications will become apparent to those skilled in the art.

CLAIMS:1. A nanoformulation of lipophilic agro waste extract with enhanced bioavailability comprising in percentage by weight;
2-20% of lipophilic agro waste extract wherein the lipophilic agro waste extract is derived from kinnow peels by SCF-CO2 extraction process;
an Organic phase having fatty acid or fatty acid derivatives in the range of 0.5-30% and optionally comprising edible oil: 0-5%; and
an aqueous dispersion phase having surfactant: 2-3%, co-surfactant: 40-60%, and remaining as water;
wherein the nanoformulation has hepatoprotective and antioxidative properties.
2. The nanoformulation according to claim 1, wherein the nanoformulation is suitable for oral administration.
3. The nanoformulation according to claim 1, wherein the fatty acid or fatty acid derivatives is selected from the group comprising stearic acid, stearic acid methyl ester and stearic acid (stearic acid ethyl ester) ethyl ester (stearic acid methyl ester), Dodecanoic acid, Myristic acid, Palmitic acid and their combination thereof.
4. The nanoformulation according to claim 1, wherein the edible oil is selected from the group comprising olive oil, canola oil, fish oil, and mixtures thereof.
5. The nanoformulation according to claim 1, wherein, the surfactants/co-surfactants are selected from the group comprising of Tween, Polysorbates, Glycerol and their combinations thereof.
6. The nanoformulation according to claim 1, wherein the kinnow peels extract comprises limonoids, flavonoids and carotenoids.
7. The nanoformulation according to claim 1, wherein the size of nanoparticle ranges from 160 to 250nm for increased aqueous dispersion and enhanced efficacy for bio-availability.
8. The use of nanoformulation comprising lipophilic agro waste extract according to claim 1, as a dietary, nutraceutical and health supplement.
9. A process of preparing a nanoformulation comprising lipophilic agro waste kinnow peels extract, the process consisting of:
a) Subjecting the kinnow peels to supercritical fluid carbon dioxide (SCF-CO2) to extract a solution rich in limonoid concentration;
b) Subjecting the kinnow peels to supercritical fluid carbon dioxide (SCF-CO2) to extract another solution rich in carotenoids and flavonoids concentration;
c) mixing the SCF-CO2 extracted kinnow peel extracts obtained in step a) and step b) to yield a solution having the required amounts of limonoids, carotenoids and flavonoids concentration wherein the amount of kinnow peels extract ranges from 0.5 to 30% (in percentage by weight) of the final formulation.
d) Stirring the solution obtained in step c) at 40oC to ensure uniform mixing and optionally adding edible oil: 0-5% in percentage by weight of the final formulation;
e) preheating the fatty acid or fatty acid derivatives (0.5-30% in percentage by weight of the final formulation) to its melting point and adding the required quantity of solution obtained in step d) to it,
f) stirring and heating the mixture obtained in step e) at 75 °C on magnetic stirrer to form a solubilized and uniform organic phase solution;
g) Adding an aqueous phase comprising surfactant: 2-3%, co-surfactant: 40-60%, and the balance of water (in percentage by weight of the final formulation) dropwise to the solution obtained in step f) under stirring conditions at 75 °C.
h) Subjecting the mixture of step (b) to Ultrasonic homogenization to obtain complete solubilization of the organic phase in the aqueous phase.
wherein the Ultrasonic homogenization is carried out with 6mm probe at 25-35% amplitude, pulse 25s on/5s off for 20 minutes at 75 °C.
10. The use of nanoformulation comprising lipophilic agro waste extract prepared according to the process of claim 9, for the treatment and/or prevention of liver diseases and has anti-oxidant benefits.