FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
COMPLETE SPECIFICATION
5 (SEE SECTION 10; RULE 13)
OMEGA FATTY ACIDS-RICH OIL DISPERSION FORMULATIONS
THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE NATURE OF THE INVENTION
AND THE MANNER IN WHICH IT IS TO BE PERFORMED
10
15
20
25
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Sr. No Name of Applicant Nationality of
Applicant
Address of the Applicant
1. BIOPLUS LIFE SCIENCES
PVT. LTD.
INDIAN 141, Walchand Hirachand Marg,
Mumbai – 400 001, Maharashtra
(IN)
2
Field of Invention:
The present invention relates to a stable and effective dispersion formulations of Omega 3 fatty
acid (O3FA)-rich oil for oral administration in infants, children’s, adult and food fortifications.
Background of Invention:
5 Docosahexanoic acid (DHA) is an essential omega fatty acid found in neuronal and other body
tissues. Proper development of neurological tissues and cognitive skills is highly dependent on
adequate intake of omega-3 fatty acids especially DHA in the diet. As the brain mass increases
approximately 3.5 times upto the age of 5 years, it requires the accumulation of Omega-3 fatty
acid (O3FA). Thus the intake of O3FA (500 mg/day) from different dietary source is highly
10 recommended during pregnancy and infancy even in adulthood by American Dietetic
Association (Kris-Etherton, PM. and Innis, S. Position of the American Dietetic Association and
Dietitian of Canada: Dietary fatty acids, J. Am. Diet Assoc., 2007, 107(9), 1599-611). Large
number of children are affected worldwide with cognitive disorder and poor neurological
conditions, Attention Deficit Hyperactivity Disorder (ADHD) is one of the most common cognitive
15 disorders of childhood. In the United States approximately 8-10% children are affected with this
disorder and in India the prevalence is even higher to 5-29%
(http://www.slideshare.net/adhdarabia/adhd-facts-and-figures). Also an increasing trend in loss
of learning disability is an alarming concern towards the cognitive health of children; about 2.4
million children are affected with this disorder in US (National center of learning disabilities,
20 2014). Omega fatty acid (O3FA) is highly lipophilic in nature. For the purpose of this
specification “Omega Fatty Acid” or “O3FA” is defined as a fatty acid which has at least one
double bond in its carbon backbone. O3FA includes, without limitation, Omega-3 fatty acid and
Omega-6 fatty acid and Omega-9 fatty acid or a combination thereof. Examples of O3FA
3
includes Docosahexaenoic acid (DHA), Eicosapantaenoic acid (EPA) Docosapantaenoic acid
(DPA) and/or Arachidonic acid; or combination thereof. Converting O3FA in order to enhance
its applicability into water soluble products is a challenging task. Presently, available formulation
like soft gelatin capsules are the most commonly used dosage form of O3FA-rich oils, which
5 suffer from their own disadvantages; like obnoxious refluxes, flatulence, low bioavailability and
the unsuitability of administration to infant and children. Moreover, the oral liquid formulations of
O3FA-rich oils are limited and even these formulations further suffers from poor dispersibility
and absorption in GIT fluid, short storage shelf life, oxidative instability and poor organoleptic
profile. Thus poor patient compliance and the lack of stable and therapeutically effective
10 formulation in children remain primary hurdles for widespread clinical use of O3FA-rich oil.
In this invention a pharmaceutically stable and organoleptically elegant O3FA-rich oil emulsion
formulation with increased bioavailability, oxidative stability, enhanced storage life and
enhanced therapeutic index has been developed. Further the safety profile is proven by cell line
studies.
15 The present invention describes an oral thixotropic emulsion in nanometric size range of O3FArich oil. The said thixotropic emulsion composition of O3FA-rich microbial oil is stable and
therapeutically efficacious. This invention also describes process of preparation of the
thixotropic emulsion composition of O3FA-rich microbial oil. The composition is optionally
fortified with vitamins and minerals.
20 O3FA in general and O3FA especially are the biomolecules implicated for proper development
of the brain and other neurological conditions. O3FA deficiency is also associated with several
medical disorders including enhanced risk of pre-term birth. But due to its highly lipophilic oily
nature, O3FA suffers from poor water dispersibility, poor organoleptic profile and oxidative
instability. Presently, only limited oral formulations of O3FA-rich oils are available under the
4
category of nutraceuticals. They possess limitations of short shelf life and poor oxidative stability
along with separation of oil during storage. In this invention, a stable thixotropic emulsion
formulation is developed for oral administration of O3FA. The stable emulsion formulation is
optionally fortified with vitamins and minerals. Therapeutics effectiveness and toxicity profile of
5 the thixotropic emulsion formulation of this invention has been demonstrated in vivo and Ex
vivo.
Emulsion is a biphasic colloidal system comprising of oil phase, water phase, emulsifiers and
stabilizers. O/W emulsion (Oil-in-water) emulsion is a best suited formulation for oral
administration of lipophilic active moieties i.e. drugs, oils, Vitamins etc., in which the oil phase is
10 well dispersed as oil globules in continuous water phase, stabilized by different emulsifiers and
stabilizers. It was surprisingly found that whereas emulsions of O3FA-rich oil are physically
unstable wherein under storage the oil separates after some time and it is also oxidatively
unstable, upon transforming the same in a thixotropic system (gel-sol-gel), the resulting
composition led to physical stability as well as oxidative stability, resulting in increased storage
15 shelf life. Further, this delivery system has flexibility to include different sweetening and flavoring
agents which increases the palatability during oral administration. Further this is easy to scale
up as the preparation described therein is simple and easy. Thus, this invention has provided a
method and a system wherein palatable and stable compositions can be done from O3FA rich
oil for nutritional therapy for health benefits brought in by O3FA.
20 DHA is a one of the most widely used O3FA plays an important role for the effective
management of pre-term birth disorder. In this invention application of developed dispersive
formulation for the treatment of pre-term birth disorder has been disclosed.
Preterm birth (<37 weeks of gestation) is one of the leading causes of infants death worldwide.
It accounts about 17% of deaths in children under 5 years of age and more than 85% of all
5
prenatal complications (Makrides M, Best K. Docosahexaenoic acid and preterm birth. Annals of
Nutrition and Metabolism. 2016;69(Suppl. 1):29-34.). Advancement in perinatal and neonatal
care will decrease the number of preterm births cases and improve cognition disorders in
infants. Epidemiological and randomized trial studies have observed an increased length of
5 gestation, infant weight and head circumference at birth in populations with high fish
consumption (Greenberg JA, Bell SJ, Van Ausdal W. Omega-3 fatty acid supplementation
during pregnancy. Reviews in obstetrics and Gynecology. 2008;1(4):162.; 3. Baack ML,
Puumala SE, Messier SE, Pritchett DK, Harris WS. What is the relationship between gestational
age and docosahexaenoic acid (DHA) and arachidonic acid (ARA) levels?. Prostaglandins,
10 Leukotrienes and Essential Fatty Acids. 2015 Sep 1;100:5-11.). Olson and Joensen first
observed that Faroe Islanders, who consume more long chain polyunsaturated fatty acids
(LCPUFA) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) rich seafood
than do Danes, had a longer gestation duration and infants with higher birth weights.
Despite enormous information about the pathophysiology of pregnant women and preterm
15 births, currently there is no strategy that can be used as a primary prevention for widespread
clinical use. The prenatal period is highly vulnerable window that is sensitive to DHA deficiency.
Omega-3 fatty acids are essential and can only be obtained from the diet. These LCPUFA
including DHA are increasingly transferred from mother to foetus late in pregnancy (3rd
trimester). Infants born before this transfer is at risk for deficiency. The requirements during
20 pregnancy have not been established, but likely exceed that of a non-pregnant state. These
fatty acids are critical for foetal neurodevelopment especially visual and neural function (Rogers
LK, Valentine CJ, Keim SA. DHA supplementation: current implications in pregnancy and
childhood. Pharmacological research. 2013 Apr 1;70(1):13-9.). According to the National
Institute of Health, the consumption of DHA is poor in developed as well as low developed
25 countries. Average intake is 40 mg in children and teens and about 90 mg in adults.
6
Recommendations for DHA are currently set at a minimum of 200-300 mg/day for pregnancy
and lactation. However, some studies have shown that supplementation with less than 600
mg/day were not beneficial in preventing early preterm birth. DHA supplementation in the range
of 600 mg-1000 mg has been associated with decreased risk of pre-term delivery and higher
5 birth weight, as well as a positive effect on infant’s brain development
(https://clinicaltrials.gov/ct2/show/NCT02626299?term=DHA+800+mg&cond=Preterm+birt
&draw=2&rank=1).
Most pregnant women do not get enough omega-3 fatty acids because the major dietary
source, seafood, is restricted to two servings a week. For pregnant women to obtain adequate
10 omega-3 fatty acids, a variety of sources should be consumed viz vegetable oils, low-mercury
fish servings a week, and supplements (fish oil or algae-based docosahexaenoic acid). Given
this emerging data, pregnant women of all dietary patterns will likely benefit from consuming a
daily DHA supplement from either fish oil or algae oil between 600-1000 mg/day. Algal oil
possess a advantage over fish oil as it is derived from microalgae, which is what fish consume
15 to get their DHA, and is biologically equivalent to the DHA available in fish oil.
Cognitive disorders are a category of mental health disorders that primarily adversely affect
learning, memory, perception, and problem solving abilities in children. These disorders range
from deep intellectual impairments to mild impairment in specific activities. These disorders
generate due to low or improper intake of O3FA during brain development phases of life. Most
20 commonly used dosage form as a source of O3FA is soft gelatin capsules of O3FA-rich oils.
but they are not suitable for administration to infants and children’s and even for adults due to
obnoxious refluxes and low bioavailability. Further limited availability and poor shelf life with
low O3FA content of liquid formulation urges a need to develop stable and therapeutically
effective formulation containing O3FA moieties.
25
7
Prior art
US Patent 2006/0165735A1 A1 discloses an oil emulsion, comprising: an oil component
comprising polyunsaturated fatty acids; an emulsifier; an emulsion stabilizer; and water; wherein
5 the oil emulsion has not been heat treated. The physical stability at room temperature and 4 C
was claimed for only 180 days. No claim for further storage was made.
US patent 2012/0251685A1 A1 discloses an oil-in-water emulsion comprising: a) an oil
containing a polyunsaturated fatty acid; b) an emulsifier; c) water; d) a metal chelating agent;
and e) an antioxidant; wherein the metal chelating agent is present in an amount from about 3%
10 to about 20% by weight of the emulsion and wherein the antioxidant is present in an amount
from about 2% to about 20% by weight of the emulsion. Storage shelf life was claimed upto five
to six months at refrigerated conditions. The use of high concentration of metal chelating agent
and antioxidants upto 20 % is considerably more than prescribed limits.
US patent 2011/0054029A1 A1 discloses a water-soluble dietary fatty acid gel formulation,
15 comprising: from 1 wt % to 75 wt % of dietary fatty acid; and from 25 wt % to 99 wt % of nonionic surfactant. Use of non-ionic surfactant hydrogenated castor oil/ macrogolglycerol
hydroxystearate (Cremophor RH 40) may have health related side effects like vasodilation,
nephrotoxicity etc when consumed at high concentration.
US 20120093998A1 discloses an emulsion comprising (i) 5-20 weight-% (wt-%), based on the
20 total weight of the emulsion, of PUFA, and (ii) 10-40 wt-%, based on the total weight of the
emulsion, of at least one emulsifier, which is a polymeric hydrocolloid originated from a plant
source, (iii) 5-45 wt-%, based on the total weight of the emulsion, of at least one adjuvant, and
(iv) 15-50 wt-%, based on the total weight of the emulsion, of water. However, no details of
globule size, physical and chemical stability of prepared formulation are given
8
US 9302017B2 discloses the micellar formulation of omega 3 fatty acid ester that from average
diameter of from about 1 μm to 10 μm. The micellar formulation was totally based on synthetic
surfactants Polysorbate 80 and Pluronic F87 using Fish oil. There was no detailed physical and
chemical stability of prepared formulation performed and strength in terms of omega 3 fatty
5 acids was also not disclosed. Inventor claimed the application of this formulation in maintenance
of cardiovascular health.
US 2011/0200644A1 discloses an emulsion comprising an emulsifier, an isotonic agent and an
oil comprising docosahexaenoic acid ethyl ester (DHA-EE), wherein the emulsion is
substantially free of eicosapentaenoic acid (EPA) and is suitable for parenteral administration,
10 The O3FA ester emulsion formation was based on Gelatin and Lipoid E80 SN. Detailed physical
and chemical stability of prepared formulation was not performed. Inventor claimed the
application of this formulation in maintenance of inflammatory conditions.
US patent 2012/0308704 A1 discloses an emulsion as an ingredient or additive for producing
food products including omega-3 fatty acids, the emulsion comprising: an outer water phase
15 including at least one water soluble antioxidant dissolved in water; and an inner fat or oil phase
which includes plant oil droplets provided with at least one fat or oil soluble antioxidant and with
an omega-3 fatty acid ester, wherein the plant oil droplets are provided with a shell made from
plant protein. The emulsion was based using Pea protein isolate as emulsifier and fish oil as a
source of omega 3 fatty acids. The developed formulation was claimed mainly for food
20 fortifications especially for production of poultry beef sausage with 1% content of omega 3 fatty
acid.
US patent 9532963B2 disclosed a food supplement or nutritional supplement composition
comprising: a fatty acid oil mixture comprising from about 25% to about 75% eicosapentaenoic
acid (EPA) and docosahexaenoic acid (DHA), by weight of the fatty acid oil mixture, wherein the
9
EPA and DHA are in a form chosen from methyl ester, ethyl ester, and triglyceride; and at least
one free fatty acid chosen from EPA, DHA, ALA, HPA, DPA, ETA, ETE, STA, linoleic acid, GLA,
AA, osbond acid, oleic acid, ricinoleic acid, erucic acid, and mixtures thereof. Formulations were
totally based on synthetic surfactants such as Cremophor, Pluronic, Briz. Inventor claimed the
5 application of this formulation in maintenance of cardiovascular health.
Thus, in none of the prior arts, problem of pre-term birth was investigated nor solved.
None of the emulsions carried a strength of DHA in more than 25 mg/ml that is required for
therapeutic efficacy for children in a couple of ml. Unless therapeutically effective dose could be
carried in reasonably small quantity of emulsion the same is not useful as practical dosage form.
10 Further, prior art compositions have synthetic surfactants, which are contraindicated for
administration in Children including neonates. Further, prior art emulsion dispersed phase is in
micrometer range, which has limited absorption of DHA. Prior art emulsions have poor stability
at room temperature. Further, preparation methods are very tedious and costly.
Hence, there was a need of improved emulsions with better and improved method of their
15 preparation.
SUMMARY OF THE INVENTION
The invention comprises an oral Omega-3-fatty acid-rich oil emulsion composition for use in
management of treatment of a disorder to overcome the same. The Omega-3-fatty acid-rich oil
may be microalge oil containing 40% DHA and the emulsion comprising DHA 50-100 mg/ml.
20 It is an embodiment of this invention that the said oral Omega-3-fatty acid-rich oil emulsion is
made from ingredients all of which are natural and biocompatible ingredients, compatible for
administration in Children including neonates
10
In one embodiment, this oral Omega-3-fatty acid-rich oil emulsion composition has the
dispersed phase is in nanometric size range.
Invention is also claimed wherein the oral Omega-3-fatty acid-rich oil emulsion composition has
stability at room temperature (about 30oC) and refrigerated one (2-8
oC).
5 In another embodiment, the oral Omega-3-fatty acid-rich oil emulsion composition comprises
Vitamins and Minerals in therapeutically effective amount.
The disorder treated by the oral Omega-3 fatty acid-rich oil emulsion comprises, without
limitation, pre-term birth disorder in pregnant women, cognitive disorders in children and
cardiovascular disorders.
10 The pre-term birth disorder in pregnant women is overcome by achieving normal delivery,
cognitive disorders in children is overcome by improvement in their cognitive ability, and a
cardiovascular disorder is overcome by return to healthy condition the oral Omega-3 fatty acidrich oil emulsion of the instant invention.
In another embodiment this invention comprises in oral Omega-3 fatty-acid-rich oil thixotropic
15 emulsion in nanomeric size range that has better absorption. The oral Omega-3-fatty-acid-rich
oil may be as a thixotropic emulsion in nanometric size range having higher surface area and
absorption. The oral Omega-3-fatty-acid-rich oil thixotropic emulsion comprises natural
emulsifier and their derivatives and bio surfactants alone or in combination with vitamins,
minerals, Generally Regarded As safe (GRAS) natural ingredients. The natural emulsifier
20 comprise, without limitation, one or more of natural gums, clays, polymers etc.; additives
comprising, without limitation, one or more selected from the group rheology modifiers, antioxidants, preservatives, stabilizers, sweetening and flavoring agents.
11
This invention also comprises an oral Omega-3 fatty-acid-rich oil emulsion composition having
dispersed phase having nanometric size comprising natural emulsifier and their derivative
surfactants alone or in combination with vitamins, minerals, Generally Regarded As safe
(GRAS) natural ingredients.
5 This invention also embodies a High Performance Liquid Chromatography (HPLC) method for
assay of omega-3 fatty acids. The method comprises steps of separately injecting blank,
standard solutions-1, standard solutions-2 and sample solution into the chromatograph,
recording the chromatograms and measuring the peak responses for Docosahexaenoic acid
(DHA). The blank injected is in a single replicate, standard solutions-1 injected are in five
10 replicates, standard solutions-2 injected are in two replicates, and sample solution injected is in
a single replicate, the column used is Thermo Syncronis C18 (250 x 4.6mm) - 5µm or
Equivalent, pump mode is isocratic, flow rate is 1.0 ml/min, detection is at UV, 210 nm, injection
volume is 20 µl, column oven temperature is 45°C and run time is 20 minutes. The solution-1
comprises DHA working standard, the solution-2 comprises DHA test solution. The sample
15 solution comprises known quantity of DHA-rich algal oil sonicated for a period of time with nHeptane in around bottom flask, Methanolic Sodium hydroxide solution is added yto the same
and refluxed for 10 minutes with a stirrer, cooled in ice bath without removing the round bottom
flask, slowly and Boron Triflouride Methanol Complex Solution is cautiously added, the solution
is refluxing further with magnetic with stirrer, cooled in ice bath without removing the round
20 bottom flask, slowly with cautiously adding n-heptane and refluxing, cooling the mixture and
removing the round bottom flask, adding saturated Sodium chloride solution, shaking well and
transferring the contents to a centrifuge tube, centrifuging with low speed, diluting upper
Heptane layer with Isopropyl Alcohol and mixing the same, further diluting this solution with
Methanol and mixing the same.
12
The natural emulsifiers comprise one or more selected from the group consisting of (i) Vitamin
E TPGS (d-α-Tocopheryl polyethylene glycol 1000 succinate); (ii) Phospholipids comprise one
or more selected from the group consisting of soya- and egg phosphatidylcholine, distearyl
phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine; gums comprise one or
5 more selected from the group consisting of gum acacia, guar gum, xanthan gum, and gum
targacanth; polymers comprise, one or more selected from the group consisting of pectin,
gelatin and alginate; emulsion stabilizers comprise one or more selected from the group
consisting of Xanthan gum, guar gum, gum acacia, Bentonite, glycerol and mixture thereof.
The oral Omega-3-fatty acid-rich oil comprise of one or more selected from the group consisting
10 of microalgae oil, fish oil or flaxseeds oil. Antioxidants comprise one or more selected from the
group consisting of butylated hydroxytoluene, rosemary oil, sodium ascorbate, Vitablend
(consisting of Vitamin E and ascorbyl palmitate), sodium metabisulphite, ascorbyl palmitate and
Vitamin E; vitamins comprise one or more selected from the group consisting of oil soluble
vitamin A, vitamin D, vitamin E, vitamin K; water soluble Vitamin B1, Vitamin B2, Vitamin B3,
15 Vitamin B5, Vitamin B6, Vitamin B12, folic acid and vitamin C. Minerals include zinc, copper,
magnesium, potassium, calcium as calcium phosphate or calcium carbonate, iron and ßcarotene etc.; buffers comprise one or more selected from the group consisting of sodium citrate
sodium carbonate and phosphate buffer flavoring agent comprise one or more flavors selected
from the group consisting of orange, strawberry, raspberry, mango, peach, vanilla, lime flavors;
20 sweetening agents comprise one or more selected from the group consisting Sorbitol, xylitol,
mannitol, Sucralose, Stevia, Aspartame, Neotame, Acesulfame K and mixtures thereof;
preservative comprises rosemary extract, sodium benzoate, sodium azide, Methyl and propyl
Paraben.
This invention also discloses a process of making oral Omega-3-fatty acid-rich oil emulsion
25 comprising steps of: (a) making an oil phase of omega-3-fatty acid-rich microalgae oil by mixing
13
a natural emulsifier, a blend of vitamins comprising an anti-oxidant with DHA-rich microalgae oil
in a manufacturing tank with stirrer at room temperature, (b) making an aqueous phase in a tank
with stirrer comprising steps of: (i) soaking a gum in purified water for a period of time required
for dissolution, (ii) dissolving Vitamin E TPGS (d-α-Tocopheryl polyethylene glycol 1000
5 succinate) in another vessel under mechanical stirring,and other water soluble ingredients
comprising a preservative and a high intensity sweetener were mixed with this solution, (iii)
thereafter, both gum and Vitamin E and Ascorbyl Palmitate solutions were mixed under
mechanical stirring for a period of time required to form uniform mixture, (iv) thereafter, oil phase
was added to the aqueous phase under mechanical stirring maintaining both phases at room
10 temperature, (v) adding flavoring agent, and continuing stirring for a further period of time.
The process of making oral Omega-3-fatty acid-rich oil emulsion according to claim 17, wherein,
the natural emulsifier comprises Soya phosphatidyl choline, the oil phase comprises a mix of: (I)
omega-3-fatty acid-rich microalgae oil 12.5 -25% w/v, (II) Anti-oxidant mix (Vitablend™ ) 0.05 to
0.5 %, (III) butylated hydroxytoluene 0.1%, (IV) DHA-rich microalgae oil 12.5 –25 % w/v, (V) the
15 Manufacturing tank is stainless steel Jacketed, (VI) stirring is done at 40-50 °C with stirring
speed of 100-300 RPM, (VII) the gum is Xanthan gum at 0.3 –1.5 % w/v), (VIII) soaking was
done in purified water at 40.-50 °C for 1-5 h period, (IX) Vitamin E TPGS (d-α-Tocopheryl
polyethylene glycol 1000 succinate) is added at 1-5 % w/v), mechanical stirring is done at
1000-1500 RPM at 1000 –1500 RPM, preservative is sodium benzoate 0.02-0.1 %w/v, (X) high
20 intensity sweetener is Sucralose 0.1 -0.5 % w/v) , (XI) uniform mixture is done by stirring and
mixing of both gum and Vitamin E TPGS solutions is done under mechanical stirring at 1000 -
1500 RPM at 40 –50 °C for 30 -60 minutes, (XII) finally, adding the oil phase was to the
aqueous phase under mechanical stirring is done at 1000 –1500 rpm, maintaining both phases
at 40–50 °C, (XIII) flavor being added is orange oil at 0.5% w/v to 1.0 %w/v and stirring
25 continued at 1 –2 hrs.
14
DETAILED DESCRIPTION OF THE INVENTION
The inventors of the present invention found that the need to develop stable and therapeutically
effective formulation containing O3FA moieties can be fulfilled by incorporation of O3FA-rich oil
into a colloidal/disperse system having nanometric size for effective absorption across the GI
5 tract. In one embodiment this invention comprises an emulsion wherein strength of DHA is 50-
100 mg/ml with normal microalge oil (contain 40% DHA).
In one embodiment of this invention that the emulsion comprises vitamins and minerals in
therapeutically effective amount.
Still further, the delivery system comprises stabilizer/s, emulsifier/s, antioxidant/s, sweetening,
10 flavoring agent/s, and vehicles.
It is also an embodiment of this invention that only natural surfactants are used. No synthetic
surfactants have been used, so the emulsion composition is good for administration in Children
including neonates
In a further embodiment of this invention, the dispersed phase of the emulsion is in a
15 nanometric range. This makes the emulsion better for absorption of DHA.
In a further embodiment, the emulsion of the instant invention has good stability at room
temperature (about 30oC and also at refrigerated temperature (about 2-8
oC).
Further, the method of preparation of the emulsion is very simple and cost effective as
compared to prior art methods of making emulsion of the DHA-rich oils, including micro algae oil
20 having 40% DHA.
O3FA-rich oil is preferably used from microalgae but may also include other O3FA-rich oil
sources such as fish oil and other sources of O3FA.
15
Examples for emulsifiers include Vitamin E TPGS (d-α-Tocopheryl polyethylene glycol 1000
succinate), phospholipids but not limited to, soya- and egg phosphatidylcholine, distearyl
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, gum acacia, guar gum,
xanthan gum, targacanth, pectin, gelatin, alginate, bentonite. The emulsifier can be defined for
5 the purpose of this invention as any molecule which possesses a Hydrophilic Lipophilic Balance
(HLB) value between 4-18.
Emulsion stabilizers can function as thickening agents, which arrest the coalescences of oil
droplets when used alone and when used in combination with rheological modifiers. Emulsion
stabilizers include, but are not limited to, Xanthan gum, guar gum, gum acacia, Bentonite,
10 glycerol and mixture thereof.
Antioxidants that prevent the autoxidation of poly unsaturated fatty acids include rosemary oil,
sodium ascorbate, Vitablend, sodium metabisulphite, ascorbyl palmitate, Vitamin E etc.
Vitamins include, but are not limited to, oil soluble vitamin A, vitamin D, vitamin E, vitamin K;
water soluble Vitamin B1, Vitamin B2, Vitamin B3, Vitamin B5, Vitamin B6, Vitamin B12, folic
15 acid and Vitamin C. Minerals include zinc, copper, magnesium, potassium, calcium as calcium
phosphate or calcium carbonate, iron and ß-carotene etc.
Buffers include, but are not limited to, sodium citrate, sodium carbonate were used to maintain
the pH of emulsion formulations.
Sweetening and flavoring agents increase the palatability of oral emulsion, they include, but are
20 not limited to orange, strawberry, raspberry, mango, peach, vanilla, lime flavors and Sorbitol,
xylitol, mannitol, Sucralose, Stevia, Aspartame, Neotame, Acesulfame K. etc. as sweeteners
used alone and mixtures thereof.
16
The stability of oral O3FA-rich oil emulsion was assessed at different storage temperature and
times points (0, 30, 60, 90 and 180 days) by assessing parameters DHA content, specific
gravity, pH, peroxide value, rheological studies, peroxide and rancimat analysis
and therapeutic effectiveness of oral O3FA-rich oil emulsion was assessed by rheological
5 studies, peroxide and rancimat analysis, in vivo cognitive animal models and ex vivo Caco2 and
Fr2 cell line studies.
Information on enhanced storage shelf life and therapeutic effectiveness of the oral O3FA-rich
oil emulsion is not available in prior art. The benefits of O3FA-rich oil emulsion formulation are
10 augmented in this invention by making an oral composition of microalgae oil emulsion that has
enhanced storage and oxidative stability. The emulsion of this invention is developed especially
in treatment of pre-term birth disorder in pregnant women and cognitive disorders in children
removed. The emulsion of this invention has shown to be effective in enhancing O3FA blood
plasma levels over prior art compositions.
15 This invention discloses a composition of O3FA-rich oil emulsion that is stable in its physical
properties and also has oxidative stability. This invention also discloses a process to prepare
pharmaceutically acceptable and stable O3FA-rich oil emulsion formulations including solid self
nano emulsifying drug delivery system, conventional emulsion, miceller dispersion alone and in
combination of vitamins and minerals with GRAS (Generally regarded as safe) certified
20 ingredients. The composition of O3FA-rich oil emulsion contains different natural emulsifiers
such as natural gums and their derivatives and bio surfactants alone or in combinations. The
composition also contains additives in optimized amount such as rheology modifiers, antioxidants, preservatives, stabilizers, sweetening and flavoring agents etc. Effect of type and
amount of emulsifier, stabilizers and antioxidants on oxidative stability, storage shelf life,
17
rheology and toxicity profile was studied by peroxide value, rheological analysis, cell line and
animal studies, respectively. The resulted emulsion has enhanced storage shelf life and is
resistant to oxidation and coalescences. The treatment of omega 3 fatty acid deficiency in
humans or animals and for reducing risk in humans associated with pre-term birth or cognitive
5 disorder or for cardiovascular health using developed formulation is disclosed. Further, new
simple, accurate and precise HPLC method for assay of major omega 3 fatty acids has been
disclosed in present invention.
This invention also embodies a novel High-performance Liquid Chromatography (HPLC)
method of analysis of DHA content in samples. Current Association of Official Agricultural
10 Chemists (AOAC) method is Gas Chromatography (GC) with Flame Ionization Detector (FID).
Related standard deviation of method of analysis with GC is 5% vs the same for inventive HPLC
method is less than 2%. GC method reproducibility is less, the inventive HPLC method
reproducibility is very good. Stability of sample solution is good in the inventive HPLC method,
poor in GC.
15 In the following are provided illustrative examples of the compositions of this invention and
investigations carried on them. The Examples are only illustrative and do not limit the scope of
the claims that would be obvious to a person skilled in the art and that would be apparent as
equivalent to a person skilled in the art.
BRIEF DESCRIPTION OF FIGURES AND LEGENDS
20 Figure 1: Illustrates the Transmission electron micrograph (TEM) of Emulsion formulation.
Figure 2: Globule size analysis of emulsion formulation using Dynamic light scattering method.
Figure 3: Illustrates the rheological analysis of O3FA-rich oil formulation (A) at constant shear
rate at 40s-1
(B) at variable shear rate 0-60s-1 (C) thixotropic analysis of 03FA- rich oil emulsion.
18
Figure 4: Illustrates the peroxide value of O3FA-rich oil emulsion A) at real time condition B) at
refrigerated conditions.
Figure 5: Illustrates the induction time at which secondary oxidation products are formed in
Rancimat analysis in emulsion (induction time 2.57h) in comparison to pure oil (induction time
5 0.45h), indicating the higher oxidative stability of emulsion.
Figure 6: Illustrates the biosafety of emulsion formulation in MTT assay against A) Caco-2 cell
line B) Fr2 cell line.
Figure 7: Illustrates A) Paracellular permeability B) TEER measurement of emulsion formulation.
(Both parameter are the measures of changes in the tight junctional and paracellular spacing of
10 gastrointestinal cells).
Figure 8: Permeability of DHA from DHA emulsion formulation in comparison to DHA oil.
Figure 9: Pharmacokinetic of optimized DHA emulsion formulation in comparison to DHA oil by
measuring DHA content in phospholipids.
Figure 10: HPLC chromatogram for method developed and validated for estimation of DHA.
15 Figure 11: Process flow diagram for commercial production of DHA emulsion.
Example 1
Preparation of a O3FA-rich oil emulsion formulations
Emulsions were prepared using O3FA- rich microalgae oil making 5-25% of the total content of
emulsion. In first step, oil phase was prepared by mixing 0.4% w/v of soya phosphatidyl choline,
20 0.05% of Vitablend, 0.1% of butylated hydroxytoluene with 12.5-25% w/v of DHA-rich
microalgae oil in stainless steel Jacketed Manufacturing tank with stirrer at 40°C with stirring
speed of 100 RPM. In second step, aqueous phase was prepared in stainless steel Jacketed
19
Manufacturing tank with stirrer. Xanthan gum (0.4% w/v) was soaked in purified water at 40°C
for 2-3 h period. In another vessel, Vitamin E TPGS (2% w/v) was dissolved under mechanical
stirring at 1000-1200 RPM at 40°C and other water soluble ingredients sodium benzoate
(0.05%w/v) and Sucralose (0.1% w/v) were mixed with this solution. Then both gum and
5 Vitamin E TPGS solutions were mixed under mechanical stirring at 1000-1200 RPM at 40°C for
30 minutes to form uniform mixture. Finally, oil phase was added to the aqueous phase under
mechanical stirring at 1000-1200 rpm, maintaining both phases at 40°C. Stirring was continued
for 0.5-1.0 hr after addition of orange oil (0.5%w/v) as flavoring agent. Process flow diagram for
commercial production of DHA emulsion in strength of 50-100 mg/ml is shown in Fig. 9.
10 Table 1: Composition of O3FA-rich oil emulsion formulation in strength 50 mg/ ml
(prototype 1)
Ingredients Quantity for 100 ml % w/v
OIL PHASE
DHA oil (40%) 12.500g 12.500%
Soy Phosphatidylcholine 0.400 g 0.400%
Ascorbyl Palmitate plus Vitamin E 0.050 g 0.050%
Butylated Hydroxytoluene 0.100 g 0.100%
Orange 5 Fold Royal Oil Flavour 0.500 g 0.500%
WATER PHASE
Xanthan gum 0.400 g 0.540%
D-α-Tocopheryl polyethylene glycol 1000
succinate (Vitamin E TPGS) 2.000 g 2.000%
Sodium Benzoate 0.050 g 0.050%
Sucralose 0.100 g 0.100%
Purified water QS for 100 ml 71.4%
20
Table 2: Composition of O3FA-rich oil emulsion formulation 75 mg/ml (prototype 2)
Ingredients Quantity for 100 ml % w/v
OIL PHASE
DHA oil (40%) 18.750g 18.750%
Soy Phosphatidylcholine 0.400 g 0.400%
Ascorbyl Palmitate plus Vitamin E 0.050 g 0.050%
Butylated Hydroxytoluene 0.100 g 0.100%
Orange 5 Fold Royal Oil Flavour 0.500 g 0.500%
WATER PHASE
Xanthan gum 0.400 g 0.400%
D-α-Tocopheryl polyethylene glycol 1000
succinate (Vitamin E TPGS) 2.000 g 2.000%
Sodium Benzoate 0.050 g 0.050%
Sucralose 0.100 g 0.100%
Purified water QS for 100 ml 77.650%
Table 3: Composition of O3FA-rich oil emulsion formulation 100 mg/ml (prototype 4)
Ingredients Quantity for 100 ml % w/v
OIL PHASE
DHA oil (40%) 18.750g 25.000%
Soy Phosphatidylcholine 0.400 g 0.400%
Ascorbyl Palmitate plus Vitamin E 0.050 g 0.050%
Butylated Hydroxytoluene 0.100 g 0.100%
Orange 5 Fold Royal Oil Flavour 0.500 g 0.500%
WATER PHASE
21
Xanthan gum 0.400 g 0.400%
D-α-Tocopheryl polyethylene glycol 1000
succinate (Vitamin E TPGS) 2.000 g 2.000%
Sodium Benzoate 0.050 g 0.050%
Sucralose 0.100 g 0.100%
Purified water QS for 100 ml 71.400%
Table 4: Composition of O3FA-rich oil emulsion formulation with Vitamins using
combination of natural emulsifiers (Prototype 4)
Ingredients Quantity for 100 ml % w/v
OIL PHASE
DHA oil (40%) 12.750g 25.000%
Soy Phosphatidylcholine 0.400 g 0.400%
Ascorbyl Palmitate plus Vitamin E 0.050 g 0.050%
Butylated Hydroxytoluene 0.100 g 0.100%
Orange 5 Fold Royal Oil Flavour 0.500 g 0.500%
WATER PHASE
Xanthan gum 0.500 g 0.400%
Pectin 0.100 g 0.100%
Bentonite 0.500 g 0.500%
D-α-Tocopheryl polyethylene glycol 1000
succinate (Vitamin E TPGS) 2.000 g 2.000%
Sodium Benzoate 0.050 g 0.050%
Sucralose 0.100 g 0.100%
Purified water QS for 100 ml 69.800%
22
Example 2: Preparation of O3FA-rich oil emulsion fortified with Vitamins and Minerals
Method of making the basic emulsion was as provided in example 1, to which Vitamins and
minerals and folic acid were added. The composition of this product has been given in Table 5
and 6.
5 Table 5: Composition of O3FA-rich oil emulsion formulation fortified with Vitamins and
Minerals
Ingredients Quantity for 100 ml % w/v
OIL PHASE
DHA oil (40%) 12.500g 12.500%
Soy Phosphatidylcholine 0.400 g 0.400%
Ascorbyl Palmitate plus Vitamin E 0.050 g 0.050%
Butylated Hydroxytoluene 0.100 g 0.100%
Orange 5 Fold Royal Oil Flavour 0.500 g 0.500%
Retinal (as vitamin A palmitate) 0.040 g 0.040%
Ergocalciferol solution 0.004 g 0.004%
WATER PHASE
Xanthan gum 0.400 g 0.400%
Thiamine hydrochloride 0.040 g 0.040%
Nicotinamide 0.800 g 0.800%
Ascorbic Acid 4.000 g 4.000%
Zinc (as zinc gluconate) 0.697 g 0.697%
Mangnese (as manganese gluconate) 0.810 g 0.810 g
D-α-Tocopheryl polyethylene glycol 1000
2.000 g 2.000%
23
succinate (Vitamin E TPGS)
Sodium Benzoate 0.050 g 0.050%
Sucralose 0.100 g 0.100%
Purified water QS for 100 ml 77.509%
Table 6: Composition of O3FA-rich oil emulsion formulation fortified with Vitamins and
Minerals
Ingredients Quantity for 100 ml % w/v
OIL PHASE
DHA oil (40%) 12.500g 12.500%
Soy Phosphatidylcholine 0.400 g 0.400%
Ascorbyl Palmitate plus Vitamin E 0.050 g 0.050%
Butylated Hydroxytoluene 0.100 g 0.100%
Orange 5 Fold Royal Oil Flavour 0.500 g 0.500%
Retinal (as vitamin A palmitate) 0.040 g 0.040%
Ergocalciferol solution 0.004 g 0.004%
WATER PHASE
Xanthan gum 0.400 g 0.400%
Pectin 0.100 g 0.100%
Bentonite 0.500 g 0.500%
Thiamine hydrochloride 0.040 g 0.040%
Nicotinamide 0.800 g 0.800%
Ascorbic Acid 4.000 g 4.000%
24
Zinc (as zinc gluconate) 0.697 g 0.697%
Mangnese (as manganese gluconate) 0.810 g 0.810%
Folic acid 0.010 g 0.010%
D-α-Tocopheryl polyethylene glycol 1000
succinate (Vitamin E TPGS) 2.000 g 2.000%
Sodium Benzoate 0.050 g 0.050%
Sucralose 0.100 g 0.100%
Purified water QS for 100 ml 76.9%
Example 3: In Vitro characterization for quality control tests
Following are the properties/characteristics studied/determined and the results are provided in
Table 7 below.
5 Globule Size and zeta potential analysis
Globule size, size distribution and zeta potential of emulsion was measured on O3FA-rich oil
emulsion prepared in Examples using particle size analyzer (Malvern Zetasizer Nano ZS90,
UK) by 100 times diluting the sample with triple distilled water. In addition to average particle
size the intensity distribution of the particles and polydispersity index (PDI) which is a measure
10 of uniformity in size distribution were also measured.
pH of Emulsion Formulations
O3FA-rich oil emulsions were developed for the oral administration; therefore the pH of the
emulsion should be within the acceptable range required for the oral administration i.e. in
between pH 5-8. The pH of the prepared emulsion formulations was determined using pH meter
15 (Toshcon CL-54).
25
Transmission electron microscopy (TEM) analysis
The size and shape of dispersed phase in the emulsion system was observed by Transmission
electron microscopy (TEM), (HRTEM, JEM 2100, JEOL, Japan) operated at an accelerating
voltage of 200 kV with beam current of 100 μA. The sample was diluted in ratio of 1:100 with
5 triple distilled water and then a drop of diluted emulsion was placed directly onto a carboncoated copper grid. The grid was air dried and was observed under transmission electron
microscope. Results are provided in Figure 1.
Thermodynamic stability study
The thermodynamic stability of the O3FA-rich oil emulsion formulations was determined by
10 storing the emulsion samples at different temperature conditions 4±1 ºC and 45±1 ºC for 24 h
storage at each temperature. Finally, after cooling and heating cycle sample was subjected to
centrifugation stress at 3000 rpm for 10 min and the extent of any oil separation was monitored.
Viscosity
The viscosity of the prepared emulsion was determined by Brookfield viscometer using spindle
15 no.5 at constant shear rate of 40 rpm/min.
Dispersibility test and robustness to dilution
Five ml of the prepared formulations were added to 500 ml of distilled water in USP Type II
Dissolution apparatus (Lab India DS 8000) at 37 ± 0.5°C and 50 rpm. The formulations were
visually examined for water dispersibility immediately after addition to the vessel using following
20 grading system: Grade A- Rapidly dispersing milky emulsion (<1 minutes); Grade B- Moderate
dispersing milky emulsion (>2 minutes); Grade C: Slow dispersion milky emulsion (>5 minutes)
with appearance of oil. Robustness to dilution is an important parameter for emulsion to ensure
that the prepared emulsion has similar properties at different dilutions after oral administration. 1
26
ml of each emulsion was diluted to 100 and 1000 times with distilled water and 0.1 N HCl. The
diluted emulsions were observed for 24 h in order to determine the separation of oily phase at
higher dilutions and globule size. Robustness to dilution is an important parameter to
understand the behaviour of emulsion globules under in vivo conditions. Separation of oily
5 phase leads to poor intestinal absorption. The results obtained are provided in Table 7.
Table 7: In vitro characterization of O3FA-rich oil emulsion formulations
Characteristic O3FA emulsion formulation O3FA emulsion
formulation fortified with
Vitamins and Minerals
Particle size (nm) after 10 fold
dilution with water
54.6 nm 146 nm
Particle size (nm) after 100
fold dilution with water
566.1 nm 612 nm
Zeta potential -17.0 mV -17.2 mV
pH 6.52 5.74
Thermodynamic stability Stable and No creaming and
no cracking.
Stable and No creaming and
no cracking
Heat cooling cycle Pass Pass
Shape Globular Globular
Viscosity (Brookfield
Viscometer spindle no. 5, 40
RPM at RT)
410 cp 990 cp
Dilution integrity
0.1 N HCl
PBS 6.8
Good (No precipitation)
Good (No precipitation)
Good (No precipitation)
Good (No precipitation)
27
PBS 7.4
Water
Good (No precipitation)
Good (No precipitation)
Good (No precipitation)
Good (No precipitation)
Dispersibility grade Grade A (Rapidly dispersing
milky emulsion (<1 minutes)
Grade A (Rapidly
dispersing milky emulsion
(<1 minutes)
Example 4
Rheological analysis
Rheological properties are important parameters for accessing the physical stability and
5 thixotropic behavior of emulsion when subjected to different shear rates and stress. Viscosity
and thixotropic profiles of O3FA emulsion was determined by using rheometer (Rheolab QC,
Anton paar, Germany). Viscosities were determined at a constant shear rate (40 s-1
) and varying
shear rate (0-60s-1
) at 25ºC temperature conditions. The thixotropic behavior of O3FA emulsion
was determined at shear rate of 80 s-1
in order to evaluate the strength recovery ratio of the
10 prepared emulsion after application and removal of shear stress. Results obtained are given in
Table 8 and Figure 3.
Table 8: Rheological behavior of emulsion formulation.
S no. Apparent Viscosity at constant
shear rate (40 s
-1
)
Thixotrophy
(%Recovery)
Breakdown
1 150- 850cp 97.0-100.25 No
28
Example 5
Peroxide value estimation
Peroxides are the primary products of oxidation formed during oxidation of oils and lipids. They
were measured according to the official method followed by Association of Analytical
5 Communities (AOAC) at time interval of 0, 30, 60 and 90 days. During this study the samples
were stored under accelerated storage conditions and refrigerated condition. Acetic acidchloroform mixture (30 mL) in the ratio 3:2 was added to the 5ml of emulsion in conical flask and
the content was properly shaken. 0.5 mL saturated solution of potassium iodide (PI) was added
and conical flask was placed in dark with occasional shaking for 1 min. 0.5 mL of 1% w/v freshly
10 prepared starch solution was added and titration was done with 0.01 N sodium thiosulphate with
vigorous shaking until blue color was disappeared. The peroxide value was calculated by using
the following equation. Results obtained are given in Figure 4.
Peroxide value (meq) = (S - B) X N (Na2S2O3) X 1000
Weight of sample
15 S = titration volume of sample
B = titration volume of blank
Example 6
Rancimat analysis
Deterioration of taste and odor (rancidity) due to oxidation is slow process at ambient
20 temperature; rancimat method accelerates the natural autooxidation process and determine the
oxidative stability of the products with time. It was determined using 892 Professional Rancimat
apparatus from Metrohm, Herisau, Switzerland at temperature condition of 90 °C with air flow
rate of 20 mL air/h. 3 ml of emulsion formulation was kept in reaction vessels which were
attached with measuring vessel and air flow pipes. Secondary metabolites formed were transfer
29
to measuring vessel with the air flow and change in the conductivity of measuring solution as
inflection point was measured. Results are provided in Figure 4.
Example 7
5 Stability study
Accelerated stability testing is going on as per ICH (International Conference for Harmonization)
guidelines by storing the O3FA-rich emulsion in sealed amber colored bottles at refrigerated
condition (2 to 8°C), and at accelerated conditions (40°C/75% RH), real time (25°C/75% RH) by
using stability chamber and results are provided in Table 9.
10 Table 9: Results of stability studies under real time and refrigeration storage conditions.
Parameters
Initial 1month 2months 3months 6 months
Storage Conditions: 25°C ± 2°C/60% ± 5% RH (Room temperature)
Appearance
Light yellow coloured
emulsion having
sweet taste and
pleasant odour.
NC NC NC NC
Physical Stability Stable Stable Stable Stable Stable
Viscosity (cP) 410 410 430 460 520
pH 5.46 5.60 5.61 5.61 5.69
Z-Average (nm)
after 100X dil 566.1
560.8 539.1 565.8 585.1
Z-Average (nm)
after 10X dil 54.6
50.5 51.7 52.2 55.7
Specific Gravity 0.9934 0.9920 0.9932 0.9961 0.9978
30
g/ml
Thixotropy (%) 99.41 99.23 97.63 96.1 94.5
Peroxide value
(meq/Kg)
1.13 1.88 1.98 2.77 2.92
Assay (% DHA
Content) 109.1 108.4 105.3 103.4 102.8
Refrigerated Storage Condition: 5°C ± 3°C
Appearance
Light yellow coloured
emulsion having
sweet taste and
pleasant odour.
NC NC NC NC
Physical Stability Stable Stable Stable Stable Stable
Viscosity (cP) 410 410 425 445 490
pH 5.46 5.65 5.65 5.69 5.72
Z-Average (nm)
after 100X dil 562.5 556.4 565.8 539.1 546.9
Z-Average (nm)
after 10X dil 49.5 42.7 52.2 55.7 55.5
Specific gravity
g/ml 0.9934 0.9967 0.9901 0.9886 0.9812
Thixotropy (%) 99.41 99.23 97.63 96.1 96.22
Peroxide value
(meq/Kg) 1.13 2.17 2.12 2.05 2.27
Assay (% DHA
Content) 109.1 105.9 104.6
104 103.8
All value is expressed as mean ± SD (n = 3). “*” represents the significant change in parameter
where p<0.05.
31
Example 8
Cell cytotoxicity assay
Cell viability assay was performed in Human colorectal (Caco2) and Normal Breast Epithelial
(Fr2) cell lines with standard MTT (3-(4,5-Dimethylthiazol-2-yl)-2,S-diphenyltetrazolium bromide)
5 method by using Human colorectal cell line (Caco2). Both the cell line were seeded in 96-well
plate and Caco2 cell line was treated with10, 20, 40, 80, 100 and 200 µM O3FA concentrations
whereas Fr2 was treated with 25, 12.5, 6.25, 3.12, 1.56 and 0.78mg/ml O3FA concentration
present in emulsion formulations for 24 hour. After O3FA-rich oil treatment, media of each well
was supplemented with 20 µL of MTT [3-(4,5-dimethylthaiazole-2-yl)-2,5 –diphenyltetrazolium
10 bromide] reagent and were further allowed to incubate for 4 h. Formed formazan crystals were
solubilized by adding 200 µL of DMSO (Dimethyl Sulphoxide) into each well. In proportion of
viable cells violet crystals were visible and absorbance was taken at 570 nm in microplate
reader (Model: Omega fluostar, BMG Labtech Ltd., Germany). The results of % cell viability are
summarized in Figure 6. and it clearly indicated that the developed DHA emulsion formulation
15 have more than 90% cell viability at all treated concentrations
Example 9
Transepithelial electrical resistance
Human colorectal cell line, (Caco-2) were cultured in Dulbecco’s modified Eagle’s medium
20 (DMEM) high Glucose supplemented with 10% FBS and 10% antibiotic-antimycotic in a
humidified incubator with 5% CO2 supply at 37 ⁰C. Cells were grown up to 60–70% confluency
and harvested with trypsin-EDTA (0.25%) and seeded at a density of 2 × 105 cells/mL onto
polycarbonate membrane Transwell inserts with pore size of 0.4 μm. Cells were cultured for 14
days to reach differentiation, and growth media was refreshed every 2–3 days. Differentiated
25 Caco-2 cells were treated with two concentrations of optimized formulation (200µg/mL) and
lipopolysaccharides (1µg/mL), taken as a positive control. After incubation period of 48 h, TEER
32
was measured using epithelial volt-ohm meter with a chopstick electrode (Millicell ERS-2, EMD
Millipore, Billerica, MA) against control. Unit area resistance was calculated by dividing the
resistance values with effective membrane area (4.52 cm2
). Observed findings are shown in
Figure 7. Treatment with developed emulsion formulations and microalgae DHA oil did not show
5 any significant change in TEER value in comparison to negative control, whereas as significant
(p<0.001) decrease in TEER value in positive control (lipopolysaccharides) was observed
(Figure 7). Thus, confirming the developed formulations did not alter the integrity of the cellular
barrier of Caco-2 cells
10 Example 10
Determination of paracellular permeability
After measurement of TEER value, treated Caco-2 cells were further analyzed for paracellular
permeability by using fluorescein isothiocyanatedextran (FD, 4 kDa). FD was dissolved in
15 phosphate buffer saline (PBS) to make concentration of 1 mg/mL. 0.2 mL of dye was added to
the apical compartment of each insert, while 1.0 mL of PBS was added to the basolateral well.
The plate was covered with foil to prevent light inactivation of the fluorescent markers and
placed in a shaker incubator at 37°C at 150 rpm. After 12 h, 0.3 mL aliquots were taken from the
basolateral chamber and fluorescence intensity was measured using a black 96 well plate in
20 multiplate reader (Fluostar Omega, BMG Lab tech, Germany). Observed findings are shown in
Figure 7
Intestinal permeation measurement
The study was performed goat ileum for developed emulsion formulation and DHA oil. With the
Tyroid solution of pH 7.4, tissue was separated and cleaned. The thread was tied to one end of
25 the ileum and placed in the student organ bath containing the Tyrode solution of pH 7.4. To
prevent peristaltic muscle contractions, the liquid solution was packed into a bag and the weight
33
of 1 g was attached to the unlined part of the lever. Aeration and bath temperatures around the
ileum bag were established at 37 ± 0.5°C. Samples were taken from the organ tube at various
intervals of 0.25, 0.5, 1, 2, 4, and 5 h, and the DHA content was measured. Results are shown
in Figure 8.
5 Pharmacokinetic study
Pharmacokinetic study was done for the period of 24 h with DHA emulsion formulation in
comparison to DHA oil and 2.2 fold increases in the absorption of DHA was observed form the
developed DHA formulation (Figure 9). This increase of DHA in plasma was depicted from DHA
content analysis by Gas Chromatography. The improved plasma levels of DHA observed during
10 pharmacokinetic study resulted from the increased permeability of DHA from intestine, which
was clearly demonstrated from the results of ex vivo intestinal permeability study.
Example 11: Development and validation of HPLC assay for Omega 3 Fatty Acids
New HPLC assay method has been developed with following chromatographic conditions have
been optimized as specified in Table 10.
15Table 10: Advantages of HPLC over GC for Analysis of DHA content in DHA-rich oil
Emulsion and Oil
S.No. Parameters HPLC Method GC Method
1 Type of Method Its non-destructive method, If the
sequence run not completed due to
errors like Instrument error, power
failure etc. the same sample
solutions can be re-injected.
It is a destructive method, If
the sequence run not completed
due to errors like Instrument
error, power failure etc. the
same sample solutions cannot
be re-injected.
34
2 Accuracy and
Precision
More precise and accurate.
RSD limit: NMT 2.0%.
Less precise and accurate
comparing to HPLC.
RSD limit: NMT 5.0%.
3 Analysis Time 20 Min (RT 11.89 Min) More comparing to HPLC
4 Solutions
stability
Stable Less stable
5 Mobile Phase Water : Methanol (950:50)
(General and Regular solvents
used in Lab and less cost)
GC Gases (Nitrogen, Hydrogen
and Zero Air). Handling of
Gases is difficult.
6 For routine
analysis
HPLC Systems Generally readily
available in Analytical laboratories
hence can be preferred.
GC Instruments generally not
readily available in Analytical
laboratories hence cannot prefer
over HPLC.
7. Column C18 (250 x 4.6mm), 5µ Thermo
Synchronis or other equivalent
column, readily available in
Analytical Laboratory.
Column cost is less comparing to
GC columns.
Dedicated column:
Fused silica capillary column
(Cyanopropyl 90%, phenyl
siloxane 10%).
More cost comparing to HPLC
columns.
8. Handling Handling is easy comparing to GC. Handling of gases is difficult in
GC analysis.
35
Chromatographic Conditions:
Column : Thermo Syncronis C18 (250 x 4.6mm), 5µm or Equivalent
Pump mode : Isocratic
5 Flow rate : 1.0 ml/min
Detection : UV, 210 nm
Injection volume : 20 µl
Column oven temperature : 45°C
Run time : 20 minutes
10 Optimized method was found to accurately measure the DHA content in experimental
protocols with high accuracy and validated for following parameters with RSD value of less than
2%. 1: Specificity and Identification, 1.1: Forced Degradation study; 2: Solution stability, 3:
Linearity, 4: Accuracy, 5: Precision - 5.1: System precision, 5.2: Method precision /
Repeatability, 5.3: Intermediate precision / Ruggedness, 6: Range, 7: Robustness - 7.1: Effect
15 of Variation in flow rate, 7.2: Effect of Variation in Wavelength of detection, 7.3: Effect of
Variation in Column oven temperature, 8: System suitability.
Preparation of Standard Solution:
Weigh accurately 250mg of DHA working standard (625mg of 40% DHA-rich microalgae oil) into
a round bottom flask, add 10mL n-Heptane by using pipette and sonicate for 15 minutes with
20 intermittent shaking. Add 20 mL of 0.5N Methanolic Sodium hydroxide solution, connect to the
condenser through Claisen adapter. Reflux the contents for 10 minutes with Magnetic Stirrer
and cool for 5minutes in ice bath without removing the round bottom flask. Slowly with
cautiously add 20 mL of Boron Triflouride Methanol Complex Solution (13-15%) through the
Claisen adapter and reflux for further 30 minutes with magnetic stirrer. Cool for 5minutes in ice
25 bath without removing the round bottom flask. Slowly with cautiously add 10ml of n-heptane
36
through Claisen adapter and reflux for further 5minutes. Cool the mixture and remove the round
bottom flask. Add 5mL of saturated Sodium chloride solution, shake well and transfer the
contents to a centrifuge tube. Centrifuge with low speed (500RPM) for 5 minutes. Dilute 2mL of
upper Heptane layer to 50mL with Isopropyl Alcohol and mix. Further dilute 5mL of this solution
5 to 50mL with Methanol and mix.
Preparation of Sample Solution: (DHA-rich oil Emulsion)
Weigh the sample accurately equivalent to 250mg of DHA-rich oil Emulsion into a round bottom
flask, add 10mL n-Heptane by using pipette and sonicate for 15 minutes with intermittent
10 shaking. In illustrative work, DHA-rich microalgae oil was used; however, any other DHA-rich oil
can be used. Add 20 mL of 0.5N Methanolic Sodium hydroxide solution, connect to the
condenser through Claisen adapter. Reflux the contents for 10 minutes with Magnetic Stirrer
and cool for 5minutes in ice bath without removing the round bottom flask. Slowly with
cautiously add 20 mL of Boron Triflouride Methanol Complex Solution (13-15%) through the
15 Claisen adapter and reflux for further 30 minutes with magnetic stirrer. Cool for 5minutes in ice
bath without removing the round bottom flask. Slowly with cautiously add 10ml of n-heptane
through Claisen adapter and reflux for further 5minutes. Cool the mixture and remove the round
bottom flask. Add 5mL of saturated Sodium chloride solution, shake well and transfer the
contents to a centrifuge tube. Centrifuge with low speed (500RPM) for 5 minutes. Dilute 2mL of
20 upper Heptane layer to 50mL with Isopropyl Alcohol and mix. Further dilute 5mL of this solution
to 50mL with Methanol and mix.
Sample Solution: (DHA-rich Oil)
Weigh the sample accurately equivalent to 250mg of DHA-rich algal oil into a round bottom
flask, add 10mL n-Heptane by using pipette and sonicate for 15 minutes with intermittent
37
shaking. Add 20 mL of 0.5N Methanolic Sodium hydroxide solution, connect to the condenser
through Claisen adapter. Reflux the contents for 10 minutes with Magnetic Stirrer and cool for
5minutes in ice bath without removing the round bottom flask. Slowly with cautiously add 20 mL
of Boron Triflouride Methanol Complex Solution (13-15%) through the Claisen adapter and
5 reflux for further 30 minutes with magnetic stirrer. Cool for 5minutes in ice bath without removing
the round bottom flask. Slowly with cautiously add 10ml of n-heptane through Claisen adapter
and reflux for further 5minutes. Cool the mixture and remove the round bottom flask. Add 5mL
of saturated Sodium chloride solution shake well and transfer the contents to a centrifuge tube.
Centrifuge with low speed (500RPM) for 5 minutes. Dilute 2mL of upper Heptane layer to 50mL
10 with Isopropyl Alcohol and mix. Further dilute 5mL of this solution to 50mL with Methanol and
mix.
Procedure:
Separately inject 20µl of solvent blank (solvent system without analyte), standard solutions-1
(containing DHA in known amount) five replicates), standard solutions-2 (containing DHA
15 sample for analysis (three replicates) and sample solution (single) into the chromatograph,
record the chromatograms and measure the peak responses for DHA.
Retention Time of DHA peak is about 11.5 minutes.
System suitability:
1) The column efficiency should be not less than 2000 theoretical plates for principal peaks.
20 2) The Theoretical plates should be not more than 2.0 for principal peaks.
3) The relative standard deviation for area response of principal peaks in five replicate injections
of standard solution should be not more than 2.0%.
4) Asymmetry (Tailing factor) in each condition is not more than 2.0.
38
Table 11: Validation of developed HPLC method for analyzing DHA
S. No.
Paramet
er
Experiment
Acceptance
criteria
Results
1. Specificit
y
Blank,
placebo,
standard
solution and
test solution.
There should
not be any
interference
due to blank,
placebo and
palmetic acid
at the
retention time
of DHA peak
There is no interference due to diluent and placebo
at the retention time of DHA peak in standard run.
The identified impurities peaks are well separated
from DHA peak.
Sample name Retention time (minutes)
Standard solution 11.87
Control sample 11.87
Peak purity
factor should
be within the
calculated
threshold limit
for DHA peak
in standard
and control
test solution
DHA Peak
Sample Purity Factor Result
Standard solution
The purity factor
is within the
calculated
threshold limit
Pass
Control test
solution
The purity factor
is within the
calculated
threshold limit
Pass
Assay of Control Sample ID % Assay
39
S. No.
Paramet
er
Experiment
Acceptance
criteria
Results
control sample sample result
should be
report
Control sample 101.4
2. Forced
degradation
Stressed
sample
No peaks
should be
detected at
the retention
time of DHA
peak in the
chromatogram
of the diluent
and placebo.
Peak purity
factor should
be within the
calculated
threshold limit
for DHA peak
DHA Peak
Sample Purity Factor Result
Control sample
The purity factor
is within the
calculated
threshold limit
Pass
Acid Degradation
The purity factor
is within the
calculated
threshold limit
Pass
Base
Degradation
The purity factor
is within the
calculated
threshold limit
Pass
40
S. No.
Paramet
er
Experiment
Acceptance
criteria
Results
in final
stressed test
solutions.
Oxidation
Degradation
The purity factor
is within the
calculated
threshold limit
Pass
3. Solution
stability
Standard
solution and
Test solution
For standard:
Recovery for
solution
stability
standard
against freshly
prepared
standard
solution
should be in
the range of
98.0% to
102.0%.
Standard Solution
Interval Area % Recovery
Initial 437770 99.7
3
rd day
5°C-8°C 23°C-27°C
99.8 99.7
For sample
solution:
Absolute
difference
Interval Area % Assay
Absolute %
Diff.
Initial 7331860 100.6
Not
Applicable
41
S. No.
Paramet
er
Experiment
Acceptance
criteria
Results
between %
assay of nth
time point and
initial time
point should
be NMT 2.0%.
3
rd day
5°C-8°C 23°C-27°C
% Assay
Absolute
% Diff.
%
Assa
y
Absol
ute %
Diff.
100.9 0.3 101.2 0.6
4. LOD and
LOQ
LOD & LOQ
Prediction
Recorded the
results
Component
Name
Concentration
(µg/ml)
Percentage
w/w
LOD LOQ LOD LOQ
0.742 2.250 1.484
4.50
0
5. Linearity Correlation
coefficient (r)
r should be
NLT 0.999
0.99973
Slope
Record the
results
148112.06011
Intercept
Record the
results
-24516.53392
% deviation of
the Yintercept
Should be not
more than ±
2.0%
-0.2
42
S. No.
Paramet
er
Experiment
Acceptance
criteria
Results
6. Accurac
y
Percentage
recovery:
Each
individual
recovery and
the mean
recovery at
each level
should be
between
98.0% -
102.0%.
Relative
standard
deviation for
% recovery of
triplicate
preparation
(each level)
should be not
more than
2.0%
% Level % Recovery Mean % RSD
Level 50% 50%
100.4
100.9 100.6 0.3
100.5
Level 100% 100%
101.6
101.7 101.6 0.1
101.6
Level 200% 200%
99.3
99.2 0.2
99.0
99.4
43
S. No.
Paramet
er
Experiment
Acceptance
criteria
Results
7. Range Method is linear of 20% to 100%, precise and accurate in the range of 50% to 200%
of test concentration.
8. Precisio
n
System
precision
Standard
solution
% RSD of DHA peak from six replicate
injections of standard
Solution-1 is NMT 2.0 %.
0.1
Method
Precision
% Assay should comply as per
specification.
Complies as per
specification
% Relative standard deviation (% RSD)
of 6 samples should be NMT 2.0%
% RSD (Method
precision)
Assay
0.6
Inter mediate
Precision
% Assay should comply as per
specification.
Complies as per
specification
% Relative standard deviation (% RSD)
of 6 samples should be NMT 2.0%
% RSD (Intermediate
precision)
Assay
0.8
44
S. No.
Paramet
er
Experiment
Acceptance
criteria
Results
Overall relative standard deviation (RSD)
of 12 samples from method precision
and intermediate precision should be not
more than 2.0%.
% RSD (Method
precision and
intermediate precision)
0.6
9. Robustn
ess
Standard
solution
System suitability should
pass.
System suitability parameters were
met as per the set acceptance
criteria
Flow rate variation
Robustness parameters were met as
per the set acceptance criteria
Wavelength variation
Column temperature Variation
45
S. No.
Paramet
er
Experiment
Acceptance
criteria
Results
10. System
suitabilit
y
Standard
solution
1. % RSD of area ratio of DHA
(Docosahexaenoic acid) peak from five
replicate injections of standard solution-1 is
not more than 2.0.
2. Calculate the % similarity factor of
standard-2 against standard-1 as per the
following formula. The % Similarity factor
should be between 98.0% - 102.0%.
3. Asymmetry (Tailing factor) for DHA
(Docosahexaenoic acid) peak is not more
than 2.0.
4. Asymmetry (Tailing factor) in each
condition is not more than 2.0.
System suitability
parameters were met
as per the set
acceptance criteria
for all the validation
parameters.
46
We Claim:
1. An oral Omega-3-fatty acid-rich oil emulsion composition for use in management of
treatment of a disorder to overcome the same.
2. The oral Omega-3-fatty acid-rich oil emulsion composition according to claim 1 wherein
5 the Omega-3-fatty acid-rich oil is microalge oil containing 40% DHA and the emulsion
comprising DHA 50-100 mg/ml.
3. The oral Omega-3-fatty acid-rich oil emulsion composition according to claim 1 wherein
all ingredients are natural and biocompatible ingredients, compatible for administration in
Children including neonates.
10 4. The oral Omega-3-fatty acid-rich oil emulsion composition according to claim 1. wherein
size of the dispersed phase is in nanometric range.
5. The oral Omega-3-fatty acid-rich oil emulsion composition according to claim 1 wherein
the same has stability at room temperature (about 30oC) and refrigerated one (2-8
oC).
6. The oral Omega-3-fatty acid-rich oil emulsion composition according to claim 1 wherein
15 the emulsion comprises Vitamins and Minerals in therapeutically effective amounts.
7. The oral Omega-3 fatty acid-rich oil emulsion composition according to claim 1, wherein
the disorder is selected from a group consisting of pre-term birth disorder in pregnant
women, cognitive disorders in children and cardiovascular disorders.
8. The oral Omega-3 fatty acid-rich oil emulsion composition according to claim 7, wherein:
20 a. pre-term birth disorder in pregnant women is overcome by achieving normal
delivery,
47
b. cognitive disorders in children is overcome by improvement in their cognitive
ability, and
c. cardiovascular disorder is overcome by return to healthy condition.
9. An oral Omega-3 fatty-acid-rich oil thixotropic emulsion in nanomeric size range and
5 having better absorption The oral Omega-3-fatty-acid-rich oil thixotropic emulsion
according to claim 4 comprising emulsion in nanometric size range having higher
surface area and absorption.
10. The oral Omega-3-fatty-acid-rich oil thixotropic emulsion according to claim 9 comprising
natural emulsifier and their derivatives and bio surfactants alone or in combination with
10 vitamins, minerals, Generally Regarded As safe (GRAS) natural ingredients.
11. The oral Omega-3-fatty-acid-rich oil thixotropic emulsion according to claim 10 wherein
the natural emulsifier comprises, one or more selected form the group consisting of
natural gums, clays and polymers.
12. The oral Omega-3-fatty-acid-rich oil thixotropic emulsion according to claim 11 wherein
15 the composition comprises additives further comprising one or more selected from the
group consisting of rheology modifiers, anti-oxidants, preservatives, stabilizers,
sweetening and flavoring agents.
13. An oral Omega-3 fatty-acid-rich oil emulsion composition having dispersed phase having
nanometric size comprising natural emulsifier and their derivative surfactants alone or in
20 combination with vitamins, minerals, Generally Regarded As safe (GRAS) natural
ingredients.
14. The oral Omega-3-fatty-acid-rich oil emulsion according to claim 13 wherein the natural
emulsifier comprises natural gums.
48
15. The oral Omega-3-fatty-acid-rich oil according to claim 13 wherein the composition
comprises additives further comprising one or more selected form the group consisting
of rheology modifiers, anti-oxidants, preservatives, stabilizers, sweetening and flavoring
agents.
5 16. A High Performance Liquid Chromatography (HPLC) method for assay of omega-3 fatty
acids.
17. The High Performance Liquid Chromatography (HPLC) method for assay of omega-3
fatty acids according to claim 11 comprising steps of:
a. separately injecting blank, standard solutions-1, standard solutions-2 and sample
10 solution into the chromatograph,
b. recording the chromatograms and
c. measuring the peak responses for Docosahexaenoic acid (DHA).
18. The High Performance Liquid Chromatography (HPLC) method for assay of omega-3
fatty acids according to claim 17, wherein: (a) the blank injected is in a single replicate,
15 (b) standard solutions-1 injected are in five replicates, (c) standard solutions-2 injected
are in two replicates, and (d) sample solution injected is in a single replicate, (e) column
used is Thermo Syncronis C18 (250 x 4.6mm), 5µm or Equivalent, (f) pump mode is
isocratic, (g) flow rate is 1.0 ml/min, (h) detection is at UV, 210 nm, (i) injection volume is
20 µl, (j) column oven temperature is 45°C, (k) run time is 20 minutes.
20 19. The High Performance Liquid Chromatography (HPLC) method for assay of omega-3
fatty acids according to claim 18, wherein:
a. the solution-1 comprises DHA working standard,
49
b. the solution-2 comprises DHA test solution,
c. sample solution comprises known quantity of DHA-rich algal oil sonicated for a
period of time with n-Heptane in around bottom flask, adding Methanolic Sodium
hydroxide solution and refluxed for 10 minutes with a stirrer, cooled in ice bath
5 without removing the round bottom flask, slowly and cautiously adding Boron
Triflouride Methanol Complex Solution, refluxing further with magnetic with
stirrer, cooled in ice bath without removing the round bottom flask, slowly with
cautiously adding n-heptane and refluxing, cooling the mixture and removing the
round bottom flask, adding saturated Sodium chloride solution, shaking well and
10 transferring the contents to a centrifuge tube. Centrifuging with low speed,
diluting upper Heptane layer with Isopropyl Alcohol and mixing the same. Further
diluting this solution with Methanol and mixing the same.
20. The oral Omega-3-fatty acid-rich oil emulsion according any one of the claims 1, claim 9,
and claim 13 wherein:
15 a. the natural emulsifiers comprise one or more selected from the group consisting
of
i. Vitamin E TPGS (d-α-Tocopheryl polyethylene glycol 1000 succinate);
ii. Phospholipids comprise one or more selected from the group consisting
of soya- and egg phosphatidylcholine, distearyl phosphatidylcholine,
20 phosphatidylethanolamine and phosphatidylserine, ;
iii. gums comprise one or more selected from the group consisting of gum
acacia, guar gum, xanthan gum, and gum targacanth, and
50
iv. polymers comprise, one or more selected from the group consisting of
pectin, gelatin and alginate,
b. emulsion stabilizers comprise one or more selected from the group consisting of
Xanthan gum, guar gum, gum acacia, Bentonite, glycerol and mixture thereof,
5 c. The oral Omega-3-fatty acid-rich oil comprise of one or more selected from the
group consisting of microalgae oil, fish oil or flaxseeds oil.
d. antioxidants comprise one or more selected from the group consisting of
butylated hydroxytoluene, rosemary oil, sodium ascorbate, Vitablend (consisting
of Vitamin E and ascorbyl palmitate), sodium metabisulphite, ascorbyl palmitate
10 and Vitamin E,
e. vitamins comprise one or more selected from the group consisting of oil soluble
vitamin A, vitamin D, vitamin E, vitamin K; water soluble Vitamin B1, Vitamin B2,
Vitamin B3, Vitamin B5, Vitamin B6, Vitamin B12, folic acid and vitamin C.
Minerals include zinc, copper, magnesium, potassium, calcium as calcium
15 phosphate or calcium carbonate, iron and ß-carotene etc.
f. buffers comprise one or more selected from the group consisting of sodium
citrate sodium carbonate and phosphate buffer flavoring agent comprise one or
more flavors selected from the group consisting of orange, strawberry, raspberry,
mango, peach, vanilla, lime flavors,
20 g. sweetening agents comprise one or more selected from the group consisting
Sorbitol, xylitol, mannitol, Sucralose, Stevia, Aspartame, Neotame, Acesulfame
K and mixtures thereof
51
h. preservative comprises rosemary extract, sodium benzoate, sodium azide,
Methyl and propyl Paraben.
21. A process of making oral Omega-3-fatty acid-rich oil emulsion comprising steps of:
a. making an oil phase of omega-3-fatty acid-rich microalgae oil by mixing a natural
5 emulsifier, a blend of vitamins comprising an anti-oxidant with DHA-rich
microalgae oil in a manufacturing tank with stirrer at room temperature
b. making an aqueous phase in a tank with stirrer comprising steps of:
i. soaking a gum in purified water for a period of time required for
dissolution,
10 ii. dissolving Vitamin E TPGS (d-α-Tocopheryl polyethylene glycol 1000
succinate) in another vessel under mechanical stirring, and other water
soluble ingredients comprising a preservative and a high intensity
sweetener were mixed with this solution,
iii. thereafter, both gum and Vitamin E and Ascorbyl Palmitate solutions were
15 mixed under mechanical stirring for a period of time required to form
uniform mixture,
iv. thereafter, oil phase was added to the aqueous phase under mechanical
stirring maintaining both phases at room temperature,
v. flavoring agent was added, and
20 vi. stirring was continued for a further period of time .
22. The oral Omega-3-fatty acid-rich oil emulsion according any one of the claims 1, claim
9, claim 13 and claim 21 wherein:
52
a. the natural emulsifiers comprise one or more selected from the group consisting
of
i. Vitamin E TPGS (d-α-Tocopheryl polyethylene glycol 1000 succinate);
ii. phospholipidstone or more selected from the group consisting of soya5 and egg phosphatidylcholine, distearyl phosphatidylcholine,
phosphatidylethanolamine and phosphatidylserine, ;
iii. gums consisting of gum acacia, guar gum, xanthan gum, and gum
targacanth, and
iv. polymers consisting of pectin, gelatin and alginate,
10 b. emulsion stabilizers comprise one or more selected from the group consisting of
Xanthan gum, guar gum, gum acacia, Bentonite, glycerol and mixture thereof,
c. antioxidants comprise one or more selected from the group consisting of
butylated hydroxytoluene, rosemary oil, sodium ascorbate, Vitablend (consisting
of Vitamin E and ascorbyl palmitate), sodium metabisulphite, ascorbyl palmitate
15 and Vitamin E,
d. vitamins comprise one or more selected from the group consisting of oil soluble
vitamin A, vitamin D, vitamin E, vitamin K; water soluble Vitamin B1, Vitamin B2,
Vitamin B3, Vitamin B5, Vitamin B6, Vitamin B12, folic acid and vitamin C.
Minerals include zinc, copper, magnesium, potassium, calcium as calcium
20 phosphate or calcium carbonate, iron and ß-carotene etc.
e. buffers comprise one or more selected from the group consisting of sodium
citrate and sodium carbonate,
53
f. flavoring agent comprise one or more flavors selected from the group consisting
of orange, strawberry, raspberry, mango, peach, vanilla, lime flavors,
g. sweetening agents comprise one or more selected from the group consisting
Sorbitol, xylitol, mannitol, Sucralose, Stevia, Aspartame, Neotame, Acesulfame
5 K and mixtures thereof,
h. preservative comprises one or more selected from the group consisting of
rosemary extract, sodium benzoate, sodium azide, Methyl and propyl Paraben ,
23. The process of making oral Omega-3-fatty acid-rich oil emulsion according to claim 17,
wherein,
10 a. the natural emulsifier comprises Soya phosphatidyl choline,
b. the oil phase comprises a mix of:
i. omega-3-fatty acid-rich microalgae oil 12.5 -25% w/v,
ii. Anti-oxidant mix (Vitablend™ ) 0.05 to 0.5 %,
iii. butylated hydroxytoluene 0.1%,
15 iv. DHA-rich microalgae oil 12.5 –25 % w/v,
v. the Manufacturing tank is stainless steel Jacketed,
vi. stirrerring is done at 40-50 °C with stirring speed of 100-300 RPM,
vii. the gum is Xanthan gum at 0.3 –1.5 % w/v),
viii. soaking was done in purified water at 40.-50 °C for 1-5 h period,
54
ix. Vitamin E TPGS (d-α-Tocopheryl polyethylene glycol 1000 succinate) is
added at 1-5 % w/v), mechanical stirring is done at 1000-1500 RPM at
1000 –1500 RPM, preservative is sodium benzoate 0.02-0.1 %w/v,
x. high intensity sweetener is Sucralose 0.1 -0.5 % w/v) ,
5 xi. uniform mixture is done by stirring and mixing of both gum and Vitamin E
TPGS solutions is done under mechanical stirring at 1000 -1500 RPM at
40 –50 °C for 30 -60 minutes .
xii. finally, adding the oil phase was to the aqueous phase under mechanical
stirring is done at 1000 –1500 rpm, maintaining both phases at 40 –50
10 °C,
xiii. flavor being added is orange oil at
0.5% w/v to 1.0 %w/v and stirring continued at 1 –2 hrs.