FIELD OF INVENTION
The present disclosure relates to the field of herbal compositions in general and micellar herbal compositions in particular. There is provided a micellar composition encapsulating fisetin.
BACKGROUND OF INVENTION
[001] Herbals possess numerous benefits and have been tapped in many products (Gurib-Fakim, Mol Aspects Med. 2006 Feb; 27(1):1-93). However, many of them come with a disadvantage of taste, bioavailability, solubility etc. (Kesarwani et al., Asian Pac J Trop Biomed. 2013 Apr; 3(4):253-66). The reduced bioavailability of herbals is one of the major limiting factors in the expanding the use of these compounds for therapeutic purposes.
[002] Natural polyphenols, and particularly the sub-group comprising flavonoids, are known and are currently being actively investigated for their antioxidant, anticancer and anti-inflammatory effects, among others. However, the bioavailability of this class of compounds remains very poor. Some of the factors causing the reduced the availability of these compounds include their low solubility, complexation to form large polymers, chelation with other compounds, and their low concentration in the source material (D’Archivio et al., Int J Mol Sci. 2010; 11(4): 1321–1342).
[003] Several strategies have been employed to increase the bioavailability of these compounds. These include loading nanoparticle formulations with these compounds, liposomal compositions, nanochelation and micellar compositions to increase the absorption of natural polyphenols. The issue that still remains is that each polyphenol has unique structural and chemical properties, and thus generic compositions aimed at increasing the absorption of polyphenols/flavonoids in general may not be effective for specific polyphenols.

[004] Thus, there is a need to develop better strategies and/or compositions to enhance the bioavailability of natural polyphenolic compounds.
[005] US5824414A describes a composition for preparing spherical polyphenol
particles.
5 [006] US 8685935 B2 is directed towards a composition that allows to increase the
bioavailability of hesperetin and consequently to maximize the beneficial health effects of hesperetin.
[007] WO2006124000A1 describes conjugating a polymer containing a free
aldehyde group with a flavonoid in the presence of an acid catalyst. The resulting
10 conjugates could be used to form delivery vehicles to deliver high doses of
flavonoids, and may also be used as delivery vehicles to deliver an additional bioactive agent.
SUMMARY OF INVENTION
[008] In an aspect of the present disclosure there is provided a method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising: (a)
15 contacting fisetin, L-α-lysophosphatidylcholine, at least one surfactant, and at least
one water soluble polymer in the presence of at least one organic solvent to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating said at least one organic solvent from said solution at a temperature in the range of 40-60°C to obtain film of micelles or clusters thereof encapsulating fisetin;
20 and (d) contacting said film with water to obtain a solution of micelles or clusters
thereof encapsulating fisetin.
[009] In an aspect of the present disclosure there is provided a composition comprising micelles or clusters thereof encapsulating fisetin prepared by a method comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, at least one
25 surfactant, and at least one water soluble polymer in the presence of at least one
organic solvent to obtain a mixture; (b) sonicating said mixture to obtain a clear and
3

transparent solution; (c) evaporating said at least one organic solvent from said
solution at a temperature in the range of 40°C -60°C to obtain film of micelles or
clusters thereof encapsulating fisetin; and (d) contacting said film with water to
obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein said
5 composition comprises: (a) fisetin encapsulated in the interior of the micelles; and (b)
the micelles, comprising: (i) L-α-lysophosphatidylcholine; (ii) at least one surfactant; and (iii) at least one water soluble polymer.
[0010] In an aspect of the present disclosure there is provided a method of increasing fisetin bioavailability in cells, said method comprising: (1) obtaining a composition
10 comprising: (a) fisetin encapsulated in the interior of the micelles; and (b) the
micelles, comprising: (i) L-α-lysophosphatidylcholine; (ii) at least one surfactant; and (iii) at least one water soluble polymer; and (2) contacting said composition with cells, wherein said composition increases fisetin bioavailability in cells. [0011] These and other features, aspects, and advantages of the present subject matter
15 will be better understood with reference to the following description and appended
claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
20 The following drawings form a part of the present specification and are included to
further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
[0012] Figure 1 shows the cellular uptake of fisetin in Caco-2 cells, in accordance
25 with an embodiment of the present disclosure.
4

[0013] Figure 2 shows the effect of micellar fisetin on LPS induced NFkB p65 ser536 phosphorylation in macrophage cells, in accordance with an embodiment of the present disclosure.
[0014] Figure 3 shows the effect of micellar fisetin on nitric oxide production in M1
5 polarized human monocytes, in accordance with an embodiment of the present
disclosure.
[0015] Figure 4 shows the effect of micellar fisetin on IL-6 production in M1
polarized human monocytes, in accordance with an embodiment of the present
disclosure.
10 [0016] Figure 5 shows the effect of micellar fisetin on IL-8 production in M1
polarized human monocytes, in accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0017] Those skilled in the art will be aware that the present disclosure is subject to
variations and modifications other than those specifically described. It is to be
15 understood that the present disclosure includes all such variations and modifications.
The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.
20 Definitions
For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and
25 known to those of skill in the art, however, for convenience and completeness,
particular terms and their meanings are set forth below.
5

[0018] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
[0019] The terms “comprise” and “comprising” are used in the inclusive, open sense,
meaning that additional elements may be included. It is not intended to be construed
5 as “consists of only”.
[0020] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.
10 [0021] The term “including” is used to mean “including but not limited to”.
“Including” and “including but not limited to” are used interchangeably.
[0022] The term micelles or clusters thereof and micellar composition are to be used interchangeably. [0023] Unless defined otherwise, all technical and scientific terms used herein have
15 the same meaning as commonly understood by one of ordinary skill in the art to
which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.
20 [0024] Fisetin (3, 3', 4', 7-tetrahydroxyflavone) belongs to the flavonoid group of
polyphenols and it occurs in fruits and vegetables such as strawberries, apples, grapes, onions and also in shrubs belonging to the family Fabaceae, such as the Acacias. Ongoing studies indicate that this compound possess anti-carcinogenic properties in addition to anti-inflammatory and antioxidant properties. Its therapeutic
25 use has been hampered, however, by its low solubility and unfavourable
pharmacokinetics.
[0025] The present disclosure provides a solution to overcome this difficulty, in the form of a micellar composition of fisetin, wherein fisetin is encapsulated in a micelle
6

comprising a surfactant, a water soluble polymer and L-α-lysophosphatidylcholine, which has demonstrably superior uptake in cells and high efficacy of inhibition of anti-inflammatory responses.
[0026] The present disclosure is not to be limited in scope by the specific
5 embodiments described herein, which are intended for the purposes of
exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.
[0027] In an embodiment of the present disclosure, there is provided a method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
10 (a) contacting fisetin, L-α-lysophosphatidylcholine, at least one surfactant, and at
least one water soluble polymer in the presence of at least one organic solvent to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating said at least one organic solvent from said solution at a temperature in the range of 40-60°C to obtain film of micelles or clusters thereof
15 encapsulating fisetin; and (d) contacting said film with water to obtain a solution of
micelles or clusters thereof encapsulating fisetin.
[0028] In an embodiment of the present disclosure, there is provided a method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, at least one surfactant, and at
20 least one water soluble polymer in the presence of at least one organic solvent to
obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating said at least one organic solvent from said solution at a temperature in the range of 40-60°C to obtain film of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with water to obtain a solution of
25 micelles or clusters thereof encapsulating fisetin, wherein said mixture is sonicated
for one cycle of five seconds at 25% power for 5 cycles.
[0029] In an embodiment of the present disclosure, there is provided a method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
7

(a) contacting fisetin, L-α-lysophosphatidylcholine, at least one surfactant, and at
least one water soluble polymer in the presence of at least one organic solvent to
obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent
solution; (c) evaporating said at least one organic solvent from said solution at a
5 temperature of 50°C to obtain film of micelles or clusters thereof encapsulating
fisetin; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof encapsulating fisetin.
[0030] In an embodiment of the present disclosure, there is provided a method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
10 (a) contacting fisetin, L-α-lysophosphatidylcholine, at least one surfactant, and at
least one water soluble polymer in the presence of at least one organic solvent to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating said at least one organic solvent from said solution at a temperature in the range of 50°C to obtain film of micelles or clusters thereof
15 encapsulating fisetin; and (d) contacting said film with water to obtain a solution of
micelles or clusters thereof encapsulating fisetin, wherein said mixture is sonicated for one cycle of five seconds at 25% power for 5 cycles.
[0031] In an embodiment of the present disclosure, there is provided a method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
20 (a) contacting fisetin, L-α-lysophosphatidylcholine, at least one surfactant, and at
least one water soluble polymer in the presence of at least one organic solvent to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating said at least one organic solvent from said solution at a temperature in the range of 40-60°C to obtain film of micelles or clusters thereof
25 encapsulating fisetin; and (d) contacting said film with water to obtain a solution of
micelles or clusters thereof encapsulating fisetin, wherein said surfactant is selected from the group consisting of sodium deoxycholate, sodium cholate, and combinations thereof.
8

[0032] In an embodiment of the present disclosure, there is provided a method of
preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
(a) contacting fisetin, L-α-lysophosphatidylcholine, at least one surfactant, and at
least one water soluble polymer in the presence of at least one organic solvent to
5 obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent
solution; (c) evaporating said at least one organic solvent from said solution at a temperature in the range of 40-60°C to obtain film of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein said surfactant is sodium
10 deoxycholate.
[0033] In an embodiment of the present disclosure, there is provided a method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, at least one surfactant, and at least one water soluble polymer in the presence of at least one organic solvent to
15 obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent
solution; (c) evaporating said at least one organic solvent from said solution at a temperature in the range of 40-60°C to obtain film of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein said at solvent is selected
20 from the group consisting of ethanol, chloroform, methanol, tertiary butanol,
trimethylamine, diethyl ether, dichloromethane, trichloromethane, cyclohexane, and combinations thereof.
[0034] In an embodiment of the present disclosure, there is provided a method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
25 (a) contacting fisetin, L-α-lysophosphatidylcholine, at least one surfactant, and at
least one water soluble polymer in the presence of at least one organic solvent to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating said at least one organic solvent from said solution at a
9

temperature in the range of 40-60°C to obtain film of micelles or clusters thereof
encapsulating fisetin; and (d) contacting said film with water to obtain a solution of
micelles or clusters thereof encapsulating fisetin, wherein said solvent is ethanol.
[0035] In an embodiment of the present disclosure, there is provided a method of
5 preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
(a) contacting fisetin, L-α-lysophosphatidylcholine, at least one surfactant, and at least one water soluble polymer in the presence of at least one organic solvent to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating said at least one organic solvent from said solution at a
10 temperature in the range of 40-60°C to obtain film of micelles or clusters thereof
encapsulating fisetin; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein said water soluble polymer is selected from the group consisting of albumin, hyaluronic acid, cellulose ethers, guar gum, carrageenan, dextran, chitosan derivatives, pectins, xanthan gum,
15 poly(ethylene glycol) (PEG), polyvinyl pyrrolidone (PVP), polyvinyl pyrrolidone
(PVP) 30, polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyacrylamides, N-(2-hydroxypropyl) methacrylamide, divinyl ether-maleic anhydride (DIVEMA), polyoxazoline, polyphosphates, and polyphosphazenes. [0036] In an embodiment of the present disclosure, there is provided a method of
20 preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
(a) contacting fisetin, L-α-lysophosphatidylcholine, at least one surfactant, and at least one water soluble polymer in the presence of at least one organic solvent to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating said at least one organic solvent from said solution at a
25 temperature in the range of 40-60°C to obtain film of micelles or clusters thereof
encapsulating fisetin; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein said water soluble polymer is polyvinyl pyrrolidone (PVP) 30.
10

[0037] In an embodiment of the present disclosure, there is provided a method of
preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
(a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and
polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b)
5 sonicating said mixture to obtain a clear and transparent solution; (c) evaporating
ethanol from said solution at a temperature in the range of 40-60°C to obtain film of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof encapsulating fisetin. [0038] In an embodiment of the present disclosure, there is provided a method of
10 preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
(a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating ethanol from said solution at a temperature in the range of 40-60°C to obtain film of
15 micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with
water to obtain a solution of micelles or clusters thereof encapsulating fisetin, , wherein said mixture is sonicated for one cycle of five seconds at 25% power for 5 cycles. [0039] In an embodiment of the present disclosure, there is provided a method of
20 preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
(a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating ethanol from said solution at a temperature of 50°C to obtain film of micelles or
25 clusters thereof encapsulating fisetin; and (d) contacting said film with water to
obtain a solution of micelles or clusters thereof encapsulating fisetin.
[0040] In an embodiment of the present disclosure, there is provided a method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
11

(a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and
polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b)
sonicating said mixture to obtain a clear and transparent solution; (c) evaporating
ethanol from said solution at a temperature in the range of 50°C to obtain film of
5 micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with
water to obtain a solution of micelles or clusters thereof encapsulating fisetin, , wherein said mixture is sonicated for one cycle of five seconds at 25% power for 5 cycles. [0041] In an embodiment of the present disclosure, there is provided a method of
10 preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
(a) contacting fisetin, L-α-lysophosphatidylcholine, at least one surfactant, and at least one water soluble polymer in the presence of at least one organic solvent to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating said at least one organic solvent from said solution at a
15 temperature in the range of 40-60°C to obtain film of micelles or clusters thereof
encapsulating fisetin; and (d) contacting said film with water to obtain a solution of
micelles or clusters thereof encapsulating fisetin, wherein L-α-
lysophosphatidylcholine has a weight percentage in said mixture in the range of 20%-40%; at least one surfactant has a weight percentage in said mixture in the range of
20 25%- 37%; at least one water soluble polymer has a weight percentage in said
mixture in the range of 25%- 37%, and fisetin has a weight percentage in said mixture in the range of 2%- 8%.
[0042] In an embodiment of the present disclosure, there is provided a method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
25 (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and
polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating ethanol from said solution at a temperature in the range of 40-60°C to obtain film of
12

micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with
water to obtain a solution of micelles or clusters thereof encapsulating fisetin,
wherein L-α-lysophosphatidylcholine has a weight percentage in said mixture in the
range of 20%-40%; sodium deoxycholate has a weight percentage in said mixture in
5 the range of 25%- 37%; polyvinylpyrrolidone K 30 has a weight percentage in said
mixture in the range of 25%- 37%, and fisetin has a weight percentage in said mixture in the range of 2%- 8%.
[0043] In an embodiment of the present disclosure, there is provided a method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
10 (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and
polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating ethanol from said solution at a temperature in the range of 40-60°C to obtain film of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with
15 water to obtain a solution of micelles or clusters thereof encapsulating fisetin,
wherein L-α-lysophosphatidylcholine has a weight percentage in said mixture of 38.89%; sodium deoxycholate has a weight percentage in said mixture of 29.17%; polyvinylpyrrolidone K 30 has a weight percentage in said mixture of 29.17%, and fisetin has a weight percentage in said mixture of 2.78%.
20 [0044] In an embodiment of the present disclosure, there is provided a method of
preparing micelles or clusters thereof encapsulating fisetin, said method comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating
25 ethanol from said solution at a temperature of 50°C to obtain film of micelles or
clusters thereof encapsulating fisetin; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein L-α-lysophosphatidylcholine has a weight percentage in said mixture of 38.89%; sodium
13

deoxycholate has a weight percentage in said mixture of 29.17%; polyvinylpyrrolidone K 30 has a weight percentage in said mixture of 29.17%, and fisetin has a weight percentage in said mixture of 2.78%.
[0045] In an embodiment of the present disclosure, there is provided a method of
5 preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
(a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating ethanol from said solution at a temperature in the range of 40-60°C to obtain film of
10 micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with
water to obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein L-α-lysophosphatidylcholine has a weight percentage in said mixture of 36.21%; sodium deoxycholate has a weight percentage in said mixture of 30.17%; polyvinylpyrrolidone K 30 has a weight percentage in said mixture of 30.17%, and
15 fisetin has a weight percentage in said mixture of 3.45%.
[0046] In an embodiment of the present disclosure, there is provided a method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b)
20 sonicating said mixture to obtain a clear and transparent solution; (c) evaporating
ethanol from said solution at a temperature in the range of 50°C to obtain film of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein L-α-lysophosphatidylcholine has a weight percentage in said mixture of
25 36.21%; sodium deoxycholate has a weight percentage in said mixture of 30.17%;
polyvinylpyrrolidone K 30 has a weight percentage in said mixture of 30.17%, and fisetin has a weight percentage in said mixture of 3.45%.
14

[0047] In an embodiment of the present disclosure, there is provided a method of
preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
(a) contacting fisetin, L-α-lysophosphatidylcholine, at least one surfactant, and at
least one water soluble polymer in the presence of at least one solvent to obtain a
5 mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c)
evaporating said at least one solvent from said solution at a temperature in the range of 40°C - 60°C to obtain film of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein said at least one surfactant to said at least one water
10 soluble polymer w/w ratio in said micelle is in the range of 1:0.83-1:1.2, and said at
least one surfactant to L-α-lysophosphatidylcholine w/w ratio in said micelle is in the range of 0.5:1.5- 1:2.
[0048] In an embodiment of the present disclosure, there is provided a method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
15 (a) contacting fisetin, L-α-lysophosphatidylcholine, at least one surfactant, and at
least one water soluble polymer in the presence of at least one solvent to obtain a mixture; (b) sonicating said mixture for one cycle of five seconds at 25% power for 5 cycles to obtain a clear and transparent solution; (c) evaporating said at least one solvent from said solution at a temperature in the range of 40°C - 60°C to obtain film
20 of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with
water to obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein said at least one surfactant to said at least one water soluble polymer w/w ratio in said micelle is in the range of 1:0.83-1:1.2, and said at least one surfactant to L-α-lysophosphatidylcholine w/w ratio in said micelle is in the range of 0.5:1.5- 1:2.
25 [0049] In an embodiment of the present disclosure, there is provided a method of
preparing micelles or clusters thereof encapsulating fisetin, said method comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b)
15

sonicating said mixture to obtain a clear and transparent solution; (c) evaporating
ethanol from said solution at a temperature in the range of 40°C - 60°C to obtain film
of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with
water to obtain a solution of micelles or clusters thereof encapsulating fisetin,
5 wherein sodium deoxycholate to polyvinylpyrrolidone K 30 w/w ratio in said micelle
is in the range of 1:0.83-1:1.2, and sodium deoxycholate to L-α-lysophosphatidylcholine w/w ratio in said micelle is in the range of 0.5:1.5- 1:2. [0050] In an embodiment of the present disclosure, there is provided a method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
10 (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and
polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b) sonicating said mixture sonicating said mixture for one cycle of five seconds at 25% power for 5 cycles to obtain a clear and transparent solution; (c) evaporating ethanol from said solution at a temperature in the range of 40°C - 60°C to obtain film of
15 micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with
water to obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein sodium deoxycholate to polyvinylpyrrolidone K 30 w/w ratio in said micelle is in the range of 1:0.83-1:1.2, and sodium deoxycholate to L-α-lysophosphatidylcholine w/w ratio in said micelle is in the range of 0.5:1.5- 1:2.
20 [0051] In an embodiment of the present disclosure, there is provided a method of
preparing micelles or clusters thereof encapsulating fisetin, said method comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating
25 ethanol from said solution at a temperature in the range of 40°C - 60°C to obtain film
of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein sodium deoxycholate to polyvinylpyrrolidone K 30 w/w ratio in said micelle
16

is 1:1, and sodium deoxycholate to L-α-lysophosphatidylcholine w/w ratio in said micelle is 1:1.33.
[0052] In an embodiment of the present disclosure, there is provided a method of
preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
5 (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and
polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating ethanol from said solution at a temperature in the range of 40°C - 60°C to obtain film of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with
10 water to obtain a solution of micelles or clusters thereof encapsulating fisetin,
wherein sodium deoxycholate to polyvinylpyrrolidone K 30 w/w ratio in said micelle is 1:1, and sodium deoxycholate to L-α-lysophosphatidylcholine w/w ratio in said micelle is 1:1.2. [0053] In an embodiment of the present disclosure, there is provided a method of
15 preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
(a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating ethanol from said solution at a temperature in the range of 40°C - 60°C to obtain film
20 of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with
water to obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein sodium deoxycholate to polyvinylpyrrolidone K 30 w/w ratio in said micelle is 1:1, and sodium deoxycholate to L-α-lysophosphatidylcholine w/w ratio in said micelle is 1:1.
25 [0054] In an embodiment of the present disclosure, there is provided a method of
preparing micelles or clusters thereof encapsulating fisetin, said method comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b)
17

sonicating said mixture to obtain a clear and transparent solution; (c) evaporating
ethanol from said solution at a temperature in the range of 40°C - 60°C to obtain film
of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with
water to obtain a solution of micelles or clusters thereof encapsulating fisetin,
5 wherein sodium deoxycholate to polyvinylpyrrolidone K 30 w/w ratio in said micelle
is 1:1, and sodium deoxycholate to L-α-lysophosphatidylcholine w/w ratio in said micelle is 1: 0.67.
[0055] In an embodiment of the present disclosure, there is provided a method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
10 (a) contacting fisetin, L-α-lysophosphatidylcholine, at least one surfactant, and at
least one water soluble polymer in the presence of at least one organic solvent to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating ethanol from said solution at a temperature in the range of 40°C - 60°C to obtain film of micelles or clusters thereof encapsulating fisetin; and
15 (d) contacting said film with water to obtain a solution of micelles or clusters thereof
encapsulating fisetin, wherein L-α-lysophosphatidylcholine has a weight percentage in said mixture in the range of 20%-40%; at least one surfactant has a weight percentage in said mixture in the range of 25%- 37%; at least one water soluble polymer has a weight percentage in said mixture in the range of 25%- 37%, and
20 fisetin has a weight percentage in said mixture in the range of 2%- 8%, wherein at
least one surfactant to at least one water soluble polymer w/w ratio in said micelle is in the range of 1:0.83-1:1.2, and at least one surfactant to L-α-lysophosphatidylcholine w/w ratio in said micelle is in the range of 0.5:1.5- 1:2. [0056] In an embodiment of the present disclosure, there is provided a method of
25 preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
(a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating
18

ethanol from said solution at a temperature in the range of 40°C - 60°C to obtain film
of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with
water to obtain a solution of micelles or clusters thereof encapsulating fisetin,
wherein L-α-lysophosphatidylcholine has a weight percentage in said mixture in the
5 range of 20%-40%; sodium deoxycholate has a weight percentage in said mixture in
the range of 25% - 37%; polyvinylpyrrolidone K 30 has a weight percentage in said mixture in the range of 25%- 37%, and fisetin has a weight percentage in said mixture in the range of 2%- 8%, wherein sodium deoxycholate to polyvinylpyrrolidone K 30 w/w ratio in said micelle is in the range of 1:0.83-1:1.2, and sodium deoxycholate to
10 L-α-lysophosphatidylcholine w/w ratio in said micelle is in the range of 0.5:1.5- 1:2.
[0057] In an embodiment of the present disclosure, there is provided a method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b)
15 sonicating said mixture to obtain a clear and transparent solution; (c) evaporating
ethanol from said solution at a temperature in the range of 40°C - 60°C to obtain film of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein L-α-lysophosphatidylcholine has a weight percentage in said mixture in the
20 range of 20%-40%; sodium deoxycholate has a weight percentage in said mixture in
the range of 25%- 37%; polyvinylpyrrolidone K 30 has a weight percentage in said mixture in the range of 25%- 37%, and fisetin has a weight percentage in said mixture in the range of 2%- 8%, wherein sodium deoxycholate to polyvinylpyrrolidone K 30 w/w ratio in said micelle is 1:1, and sodium deoxycholate to L-α-
25 lysophosphatidylcholine w/w ratio in said micelle is 1:1.33.
[0058] In an embodiment of the present disclosure, there is provided a method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and
19

polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b)
sonicating said mixture to obtain a clear and transparent solution; (c) evaporating
ethanol from said solution at a temperature in the range of 40°C - 60°C to obtain film
of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with
5 water to obtain a solution of micelles or clusters thereof encapsulating fisetin,
wherein L-α-lysophosphatidylcholine has a weight percentage in said mixture of 38.89%; sodium deoxycholate has a weight percentage in said mixture of 29.17%; polyvinylpyrrolidone K 30 has a weight percentage in said mixture of 29.17%, and fisetin has a weight percentage in said mixture of 2.78%, wherein sodium
10 deoxycholate to polyvinylpyrrolidone K 30 w/w ratio in said micelle is 1:1, and
sodium deoxycholate to L-α-lysophosphatidylcholine w/w ratio in said micelle is 1:1.33.
[0059] In an embodiment of the present disclosure, there is provided a method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
15 (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium deoxycholate, and
polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating ethanol from said solution at a temperature in the range of 40°C - 60°C to obtain film of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with
20 water to obtain a solution of micelles or clusters thereof encapsulating fisetin,
wherein L-α-lysophosphatidylcholine has a weight percentage in said mixture of 36.21%; sodium deoxycholate has a weight percentage in said mixture of 30.17%; polyvinylpyrrolidone K 30 has a weight percentage in said mixture of 30.17%, and fisetin has a weight percentage in said mixture of 3.45% wherein sodium
25 deoxycholate to polyvinylpyrrolidone K 30 w/w ratio in said micelle is 1:1, and
sodium deoxycholate to L-α-lysophosphatidylcholine w/w ratio in said micelle is 1:1.2.
20

[0060] In an embodiment of the present disclosure, there is provided a composition
wherein: (a) fisetin is encapsulated in the interior of the micelles; and (b) the
micelles comprise: (i) L-α-lysophosphatidylcholine; (ii) at least one surfactant; and
(iii) at least one water soluble polymer, wherein in said composition is prepared by a
5 method comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, said at least
one surfactant, and said at least one water soluble polymer in the presence of at least one organic solvent to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating ethanol from said solution at a temperature in the range of 40°C - 60°C to obtain film of micelles or clusters thereof
10 encapsulating fisetin; and (d) contacting said film with water to obtain a solution of
micelles or clusters thereof encapsulating fisetin.
[0061] In an embodiment of the present disclosure, there is provided a composition wherein: (a) fisetin is encapsulated in the interior of the micelles; and (b) the micelles comprise: (i) L-α-lysophosphatidylcholine; (ii) at least one surfactant; and (iii) at least
15 one water soluble polymer, wherein in said composition is prepared by a method
comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, at least one surfactant, and at least one water soluble polymer in the presence of at least one organic solvent to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating ethanol from said solution at a temperature in the
20 range of 40°C - 60°C to obtain film of micelles or clusters thereof encapsulating
fisetin; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein at least one surfactant to at least one water soluble polymer w/w ratio in said micelle is in the range of 1:0.83-1:1.2, and at least one surfactant to L-α-lysophosphatidylcholine w/w ratio in said micelle is in the
25 range of 0.5:1.5- 1:2.
[0062] In an embodiment of the present disclosure, there is provided a composition wherein: (a) fisetin is encapsulated in the interior of the micelles; and (b) the micelles comprise: (i) L-α-lysophosphatidylcholine; (ii) at least one surfactant; and (iii) at least
21

one water soluble polymer, wherein said composition is prepared by a method
comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, said at least one
surfactant, and said at least one water soluble polymer in the presence of at least one
organic solvent to obtain a mixture; (b) sonicating said mixture to obtain a clear and
5 transparent solution; (c) evaporating said at least one organic solvent from said
solution at a temperature in the range of 40°C - 60°C to obtain film of micelles or
clusters thereof encapsulating fisetin; and (d) contacting said film with water to
obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein L-α-
lysophosphatidylcholine has a weight percentage in said mixture in the range of 20%-
10 40%; at least one surfactant has a weight percentage in said mixture in the range of
25%- 37%; at least one water soluble polymer has a weight percentage in said
mixture in the range of 25%- 37%, and fisetin has a weight percentage in said mixture
in the range of 2%- 8%, wherein at least one surfactant to at least one water soluble
polymer w/w ratio in said micelle is in the range of 1:0.83-1:1.2, and at least one
15 surfactant to L-α-lysophosphatidylcholine w/w ratio in said micelle is in the range of
0.5:1.5- 1:2.
[0063] In an embodiment of the present disclosure, there is provided a composition
wherein: (a) fisetin is encapsulated in the interior of the micelles; and (b) the micelles
comprise: (i) L-α-lysophosphatidylcholine; (ii) at least one surfactant; and (iii) at least
20 one water soluble polymer, wherein said at least one surfactant in said composition is
sodium deoxycholate; said at least one water soluble polymer in said composition is polyvinylpyrrolidone K 30; and said at least one solvent is ethanol.
[0064] In an embodiment of the present disclosure, there is provided a composition
wherein: (a) fisetin is encapsulated in the interior of the micelles; and (b) the micelles
25 comprise: (i) L-α-lysophosphatidylcholine; (ii) sodium deoxycholate; and (iii) at least
polyvinylpyrrolidone K 30, wherein said composition is prepared by a method
comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium
deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a
22

mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c)
evaporating ethanol from said solution at a temperature in the range of 40°C - 60°C to
obtain film of micelles or clusters thereof encapsulating fisetin; and (d) contacting
said film with water to obtain a solution of micelles or clusters thereof encapsulating
5 fisetin, wherein at sodium deoxycholate to polyvinylpyrrolidone K 30 w/w ratio in
said micelle is in the range of 1:0.83-1:1.2, and sodium deoxycholate to L-α-lysophosphatidylcholine w/w ratio in said micelle is in the range of 0.5:1.5- 1:2. [0065] In an embodiment of the present disclosure, there is provided a composition wherein: (a) fisetin is encapsulated in the interior of the micelles; and (b) the micelles
10 comprise: (i) L-α-lysophosphatidylcholine; (ii) sodium deoxycholate; and (iii) at least
polyvinylpyrrolidone K 30, wherein in said composition is prepared by a method
comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium
deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c)
15 evaporating ethanol from said solution at a temperature in the range of 40°C - 60°C to
obtain film of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein sodium deoxycholate to polyvinylpyrrolidone K 30 w/w ratio in said micelle is 1:1, and sodium deoxycholate to L-α-lysophosphatidylcholine w/w ratio in
20 said micelle is 1:1.33.
[0066] In an embodiment of the present disclosure, there is provided a composition wherein: (a) fisetin is encapsulated in the interior of the micelles; and (b) the micelles comprise: (i) L-α-lysophosphatidylcholine; (ii) sodium deoxycholate; and (iii) at least polyvinylpyrrolidone K 30, wherein in said composition is prepared by a method
25 comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium
deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating ethanol from said solution at a temperature in the range of 40°C - 60°C to
23

obtain film of micelles or clusters thereof encapsulating fisetin; and (d) contacting
said film with water to obtain a solution of micelles or clusters thereof encapsulating
fisetin, wherein sodium deoxycholate to polyvinylpyrrolidone K 30 w/w ratio in said
micelle is 1:1, and sodium deoxycholate to L-α-lysophosphatidylcholine w/w ratio in
5 said micelle is 1:1.2.
[0067] In an embodiment of the present disclosure, there is provided a composition wherein: (a) fisetin is encapsulated in the interior of the micelles; and (b) the micelles comprise: (i) L-α-lysophosphatidylcholine; (ii) sodium deoxycholate; and (iii) at least polyvinylpyrrolidone K 30, wherein said composition is prepared by a method
10 comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium
deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c) evaporating ethanol from said solution at a temperature in the range of 40°C - 60°C to obtain film of micelles or clusters thereof encapsulating fisetin; and (d) contacting
15 said film with water to obtain a solution of micelles or clusters thereof encapsulating
fisetin, wherein sodium deoxycholate to polyvinylpyrrolidone K 30 w/w ratio in said micelle is 1:1, and sodium deoxycholate to L-α-lysophosphatidylcholine w/w ratio in said micelle is 1:1. [0068] In an embodiment of the present disclosure, there is provided a composition
20 wherein: (a) fisetin is encapsulated in the interior of the micelles; and (b) the micelles
comprise: (i) L-α-lysophosphatidylcholine; (ii) sodium deoxycholate; and (iii) at least
polyvinylpyrrolidone K 30, wherein in said composition is prepared by a method
comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium
deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a
25 mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c)
evaporating ethanol from said solution at a temperature in the range of 40°C - 60°C to obtain film of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof encapsulating
24

fisetin, wherein sodium deoxycholate to polyvinylpyrrolidone K 30 w/w ratio in said micelle is 1:1, and sodium deoxycholate to L-α-lysophosphatidylcholine w/w ratio in said micelle is 1:0.67.
[0069] In an embodiment of the present disclosure, there is provided a composition
5 wherein: (a) fisetin is encapsulated in the interior of the micelles; and (b) the micelles
comprise: (i) L-α-lysophosphatidylcholine; (ii) sodium deoxycholate ; and (iii)
polyvinylpyrrolidone K 30, wherein said composition is prepared by a method
comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium
deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a
10 mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c)
evaporating ethanol from said solution at a temperature in the range of 40°C - 60°C to obtain film of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein L-α-lysophosphatidylcholine has a weight percentage in said mixture
15 in the range of 20%-40%; sodium deoxycholate has a weight percentage in said
mixture in the range of 25%- 37%; polyvinylpyrrolidone K 30 has a weight percentage in said mixture in the range of 25%- 37%, and fisetin has a weight percentage in said mixture in the range of 2%- 8%, wherein sodium deoxycholate to polyvinylpyrrolidone K 30 w/w ratio in said micelle is in the range of 1:0.83-1:1.2,
20 and sodium deoxycholate to L-α-lysophosphatidylcholine w/w ratio in said micelle is
in the range of 0.5:1.5- 1:2.
[0070] In an embodiment of the present disclosure, there is provided a composition wherein: (a) fisetin is encapsulated in the interior of the micelles; and (b) the micelles comprise: (i) L-α-lysophosphatidylcholine; (ii) sodium deoxycholate ; and (iii)
25 polyvinylpyrrolidone K 30, wherein said composition is prepared by a method
comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium
deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c)
25

evaporating ethanol from said solution at a temperature in the range of 40°C - 60°C to
obtain film of micelles or clusters thereof encapsulating fisetin; and (d) contacting
said film with water to obtain a solution of micelles or clusters thereof encapsulating
fisetin, wherein L-α-lysophosphatidylcholine has a weight percentage in said mixture
5 of 38.89%; sodium deoxycholate has a weight percentage in said mixture of 29.17%;
polyvinylpyrrolidone K 30 has a weight percentage in said mixture of 29.17%, and fisetin has a weight percentage in said mixture of 2.78%.
[0071] In an embodiment of the present disclosure, there is provided a composition wherein: (a) fisetin is encapsulated in the interior of the micelles; and (b) the micelles
10 comprise: (i) L-α-lysophosphatidylcholine; (ii) sodium deoxycholate ; and (iii)
polyvinylpyrrolidone K 30, wherein said composition is prepared by a method
comprising: (a) contacting fisetin, L-α-lysophosphatidylcholine, sodium
deoxycholate, and polyvinylpyrrolidone K 30 in the presence of ethanol to obtain a mixture; (b) sonicating said mixture to obtain a clear and transparent solution; (c)
15 evaporating ethanol from said solution at a temperature in the range of 40°C - 60°C to
obtain film of micelles or clusters thereof encapsulating fisetin; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof encapsulating fisetin, wherein L-α-lysophosphatidylcholine has a weight percentage in said mixture of 3.45%; sodium deoxycholate has a weight percentage in said mixture of 36.21%;
20 polyvinylpyrrolidone K 30 has a weight percentage in said mixture of 36.21%, and
fisetin has a weight percentage in said mixture of 3.45%.
[0072] In an embodiment of the present disclosure, there is provided a method of increasing fisetin bioavailability in cells, said method comprising: (a) obtaining a composition comprising: (a) fisetin encapsulated in the interior of micelles; and (b)
25 micelles comprising: (i) L-α-lysophosphatidylcholine; (ii) sodium deoxycholate; and
(iii) polyvinylpyrrolidone K 30; and (b) contacting said micelles or clusters thereof with cells, wherein said method increases fisetin bioavailability in cells.
26

[0073] In an embodiment of the present disclosure, there is provided a method of
increasing fisetin bioavailability in cells, said method comprising: (a) obtaining a
composition comprising: (a) fisetin encapsulated in the interior of micelles; and (b)
micelles comprising: (i) L-α-lysophosphatidylcholine; (ii) sodium deoxycholate; and
5 (iii) polyvinylpyrrolidone K 30; wherein sodium deoxycholate to
polyvinylpyrrolidone K 30 w/w ratio in said micelle is in the range of 1:0.83-1:1.2, and sodium deoxycholate to L-α-lysophosphatidylcholine w/w ratio in said micelle is in the range of 0.5:1.5- 1:2; and (b) contacting said micelles or clusters thereof with cells, wherein said method increases fisetin bioavailability in cells.
10 [0074] In an embodiment of the present disclosure, there is provided a method of
increasing fisetin bioavailability in cells, said method comprising: (a) obtaining a
composition comprising: (a) fisetin encapsulated in the interior of micelles; and (b)
micelles comprising: (i) L-α-lysophosphatidylcholine; (ii) sodium deoxycholate; and
(iii) polyvinylpyrrolidone K 30; wherein sodium deoxycholate to
15 polyvinylpyrrolidone K 30 w/w ratio in said micelle is 1:1, and sodium deoxycholate
to L-α-lysophosphatidylcholine w/w ratio in said micelle is 1:1.33; and (b) contacting said micelles or clusters thereof with cells, wherein said method increases fisetin bioavailability in cells. [0075] In an embodiment of the present disclosure, there is provided a method of
20 increasing fisetin bioavailability in cells, said method comprising: (a) obtaining a
composition comprising: (a) fisetin encapsulated in the interior of micelles; and (b)
micelles comprising: (i) L-α-lysophosphatidylcholine; (ii) sodium deoxycholate; and
(iii) polyvinylpyrrolidone K 30; wherein sodium deoxycholate to
polyvinylpyrrolidone K 30 w/w ratio in said micelle is 1:1, and sodium deoxycholate
25 to L-α-lysophosphatidylcholine w/w ratio in said micelle is 1:1.2; and (b) contacting
said micelles or clusters thereof with cells, wherein said method increases fisetin bioavailability in cells
27

[0076] In an embodiment of the present disclosure, there is provided micelles or clusters thereof encapsulating fisetin as described herein for use in preparing formulations for oral ingestion.
[0077] In an embodiment of the present disclosure, there is provided micelles or
5 clusters thereof as described herein, further comprising suitable food grade or
cosmetic grade additives.
[0078] In an embodiment of the present disclosure, there is provided micelles or clusters thereof further comprising suitable additives, wherein said additives are substances which when added in small quantities help to improve or preserve said
10 micelles.
[0079] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art
15 upon reference to the description of the subject matter. It is therefore contemplated
that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.
EXAMPLES
The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any
20 limitations on the scope of the present disclosure. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary
25 methods, devices and materials are described herein. It is to be understood that this
28

disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.
Example 1
Material and Methods
5 [0080] Cell culture and reagents: Mouse leukemic macrophage cell line, RAW 264.7
(ATCC-TIB 71), and Caco-2, human epithelial colorectal adenocarcinoma cells (ATCC HTB-37) (American Type Culture Collection Rockville, MD, USA) and cells were cultured in complete Dulbecco's Modified Eagle's medium (DMEM) (Sigma Aldrich) with 10% fetal bovine serum (FBS) (Gibco). The human monocytic cell line
10 U937 was cultured in Roswell Park Memorial Institute medium (RPMI) 1640
medium (Sigma Aldrich) supplemented with 10% (v/v) FBS, 2 mM glutamine, 100
U/mL penicillin and 100 μg/mL streptomycin at 37°C under 5% CO2. L-α-
lysophosphatidylcholine (62963), sodium deoxycholate (D6750),
polyvinylpyrrolidone K 30 (81420), fisetin (F4043) and phorbol 12-myristate 13-
15 acetate (P8139) and Griess’ reagent for nitrite (03553) were purchased from Sigma-
Aldrich.
[0081] Thin Film Dispersion Method: The fisetin loaded micelles were prepared using thin-film dispersion method. Briefly, an appropriate amount of fisetin (2.86 mg), L-α-lysophosphatidylcholine (40-10 mg), sodium deoxycholate (30-15 mg) and
20 polyvinylpyrrolidone K 30 (30-15 mg) were dissolved in 2 ml of ethanol with
ultrasonic treatment (sonication with one cycle of five seconds at 25% power for 5 cycles) to form a clear and transparent solution. The organic solvent was then evaporated by incubating at 50º C to obtain a film of fisetin loaded micelle mixture. Subsequently, this was dissolved with in double distilled water. The final micellar
25 solution was stored at 4º C for further analysis.
29

[0082] Four different micellar compositions were prepared using the thin film dispersion method. The weight range used for the different components in these micellar preparations are listed in Table 1.
30

Table 1

Ingredients Micellar
Preparation
1 Micellar
Preparation
2 Micellar
Preparation
3 Micellar
Preparation
4
Fisetin 2.86 mg 2.86 mg 2.86 mg 2.86 mg
L-α-lysophosphatidylcholine 40 mg 30 mg 20 mg 10 mg
Sodium deoxycholate 30 mg 25 mg 20 mg 15 mg
Polyvinylpyrrolidone K 30 30 mg 25 mg 20 mg 15 mg
Ethanol 2 ml 2 ml 2 ml 2 ml
[0083] Cellular uptake of fisetin in Caco-2 cells: Caco-2 cells were seeded at a
density of 5 × 105 cells per well. The cells were grown in DMEM medium
5 supplemented with 1% non-essential amino acids, 1% of penicillin-streptomycin and
10% FBS. The medium was changed every alternate day. Cellular uptake studies were carried out with cell monolayers that were 21 days old. For each treatment, two wells of cells were used for each sample, and each treatment was repeated in triplicate. Caco-2 cells were treated with free fisetin and micellar fisetin for 6 hours.
10 After the treatment the cells were washed thrice in 1X PBS for 5 minutes each.
Immediately post-wash, the cells were visualized using LSM 710 confocal microscope (Carl Zeiss) using appropriate laser, filter settings and 40x oil immersion objective. Quantification analysis of the fisetin auto-fluorescence intensity was performed at various depth of the cells using Zeiss 2010 analysis software.
15 [0084] Macrophage differentiation and polarization of U937 cells: Polarization of
U937 cells were differentiated to macrophages with phorbol-12-myristate-13-acetate (PMA). U937 cells were cultured for three days in complete RPMI with or without 50 nM PMA. Upon treatment with PMA, cells underwent growth arrest and a change of
31

morphology owing to their attachment to the substrate. The cells were treated with 25
mg/ml of LPS for the last 12 h of PMA. To confirm the macrophage polarization, the
amount levels of the M1 polarization markers like IL6, IL-8 and nitric oxide were
analyzed in the supernatant with the specific ELISA kits, according to the
5 manufacturer’s instructions.
Example 2
Fisetin micelles show enhanced cellular uptake
[0085] Free fisetin and the fisetin micellar compositions were administered to cells as
10 described in the protocol above. As seen in Figure 1, all four micellar compositions of
fisetin showed superior uptake in cells as compared to fisetin alone. Among the four preparations, preparation 1 at 32.9% uptake, followed by preparation 2 and 3 at 24.76% and 20.3% uptake, respectively showed the highest bioavailability. Preparation 4 had the least uptake of fisetin among the preparations showing a 2.65%
15 uptake as compared to fisetin alone. Thus, it can be inferred from this data that all the
micellar compositions tried, enhance the bioavailability of fisetin, as compared to free fisetin, and preparation 1 is the most efficacious among the compositions tested.
Example 3
20 Fisetin micelles show enhanced inhibition on of LPS induced NFkB p65 ser536
phosphorylation
[0086] Fisetin is known to function as an antioxidant and is reputed to have anti-carcinogenic and anti-inflammatory effects, however its poor bioavailability has made it difficult to elucidate the properties of this molecule further.
25 [0087] In order to test whether the fisetin micelle preparations could promote anti-
inflammatory responses, their effect was studied on LPS induced NFkB p65 ser536 phosphorylation. The NFkB group of proteins are expressed in M1 macrophages as a common response to microbial infection. They are produced in response to bacterial
32

antigens and surface molecules, such as lipopolysaccharides (LPS). The assay was performed in the manner described below.
[0088] RAW macrophage cells were seeded into each well of 12-well plates until
80% confluent. NFκB p65 ser 536 phosphorylation was stimulated with (10 μg/ml)
5 lipopolysaccharides (LPS). This treatment induces the inflammatory response within
90 minutes of stimulation. Simultaneously, the cells were treated with free fisetin and micellar fisetin. Detection of NFκB p65 ser 536 phosphorylation was done using sandwich ELISA (Cell Signalling Inc). Briefly, media was removed from the cells and the cells were rinsed with ice cold 0.1M phosphate buffer saline (1X). Cells were
10 lysed with 0.5 mL ice cold 1X cell lysis buffer (20mM Tris-HCl (pH 7.5), 150mM
NaCl, 1mM Na2EDTA, 1mM EGTA, 1% triton, 2.5mM sodium pyrophosphate, 1mM beta-glycerophosphate, 1mM Na3VO4, 1µg/mL leupeptin, 1mM PMSF (phenylmethylsulfonyl fluoride) (immediately before use) and incubated on ice for five minutes. The cells were subsequently scraped from the plate and transferred to a
15 tube and lysed using probe sonication (sonication with one cycle of five seconds at
25% power, samples kept on ice during sonication to prevent excessive heating). The lysate was then spun at 14,000 RPM for 10 minutes at 4ºC and transferred to a new tube. Cell lysates when needed, were diluted with sample diluents (supplied with kit). The lysates were then transferred to 96 well plates and incubated overnight at 37ºC.
20 Next, washing of the wells was done with 1X wash buffer supplied with the kit
[PathScan® Phospho-NF-κB p65 (Ser536) Sandwich ELISA Kit (Cell Signaling Technology, USA; Cat # 7173C]. To each well, 100μL of NF-κB p65 detection antibody (supplied with kit) was added and the plate was incubated at 37ºC for one-hour. The wells were then washed again and 100 µL of horseradish peroxidase
25 (HRP) -linked secondary antibody was added to each well. The plate was then
incubated for 30 minutes at 37°C. The wash step was repeated and 100 µL of 3, 3′, 5, 5′-tetramethylbenzidine (TMB) buffer was added. Finally, the stop solution was added and absorbance read at 450 nm.
33

[0089] As seen in Figure 2, micellar preparation 1 and preparation 2 showed higher
percentage inhibition as compared to preparations 3 and 4. The percentage inhibition
demonstrated by preparation 1 was at 26.28%, which is close to two-fold higher than
what is observed by administering fisetin alone. Preparation 2 also showed high
5 percentage inhibition at 21.55%.
Example 4
Fisetin micelles show enhanced inhibition on of LPS induced Nitric Oxide
production
10 [0090] In response to environmental stressors such as cellular damage and microbial
infection, monocytes undergo polarization to form the M1 subtype of macrophages. Following polarization, they exhibit a suite of cellular responses, including production of pro-inflammatory cytokines and production of Nitric Oxide (NO) and reactive oxygen species. The efficacy of the fisetin micelles in inhibiting the
15 production of NO was tested using the protocol below.
[0091] Polarized M1 cells were treated with free fisetin and micellar fisetin for 24 hours. The concentration of NO in culture supernatants was determined as nitrite using Griess reagent. Briefly, 100 μL of cell culture medium with an equal volume of Griess reagent in a 96-well plate was incubated at room temperature for 10 min. Then
20 the absorbance was measured at 540 nm in a microplate reader (Thermo Fisher,
Germany). The amount of nitrite in the media was calculated from sodium nitrite (NaNO2) standard curve.
[0092] As seen in Figure 3, micellar preparation 1 was the most efficacious among all the preparations in inhibiting NO production. At 52.38% inhibition, the level of
25 inhibition demonstrated by this preparation was close to the inhibitory levels
demonstrated by the positive control curcumin. Fisetin micellar preparation 1 also showed an almost two-fold increase in inhibition of NO levels as compared to fisetin alone. At this instance, the other preparations did not show levels percentage
34

inhibition values that were significantly different than fisetin alone. Thus, micellar preparation 1 shows enhanced efficacy in treating inflammatory responses.
Example 5
5 Fisetin micelles show enhanced inhibition on LPS induced IL-6 and IL-8
production
[0093] Pro-inflammatory responses include the production of cytokines such as Interleukin-6 (IL-6) and Interleukin-8 (IL-8). These chemokines also produced in the defence responses of M1 polarized macrophages. The inhibition of IL-6 and IL-8
10 production by fisetin micelles was tested by treating polarized M1 cells with free
fisetin and micellar fisetin for 24 hours. To quantify IL-6 and IL-8 production, culture supernatants were collected and the levels of secreted cytokines were determined using an ELISA kit (Krishgen BioSystems, India). [0094] As seen in Figures 4 and 5, among the fisetin micelle preparations,
15 preparation 1 was the most effective among the micellar preparations in inhibiting IL-
6 and IL-8 production.
[0095] Figure 4 shows that micellar preparation 1 can inhibit IL-6 production by 39.29% which is close to the levels of inhibition by the positive control curcumin at 40.60%. Micellar preparation 1 is also 1.5 times fold more efficacious than fisetin
20 alone in inhibiting IL-6 levels.
[0096] The percentage inhibition of IL-8 by preparation 1 was found to be 65.73%, which is close to the levels of inhibition demonstrated by the positive control curcumin, and represents an approximately 34% increase over the percentage inhibition showed by fisetin alone (Figure 5). While other micellar preparations were
25 not effective in inhibiting IL-6 levels as compared to fisetin, preparation 2 and 3 were
moderately effective in inhibiting IL-8 production.
[0097] Overall, these data suggest that fisetin bioavailability is quite poor when administered to cells by itself. Encapsulating fisetin in micelles provides a solution to
35

help overcome the problem of fisetin bioavailability. It can be further appreciated
from the instant disclosure, that while micellar encapsulation is necessary to increase
fisetin bioavailability, some micellar preparations at particular w/w ratios are more
efficacious than others. Only a particular composition of micelle, wherein sodium
5 deoxycholate to polyvinylpyrrolidone K 30 w/w ratio in said micelle is 1:1, and
sodium deoxycholate to L-α-lysophosphatidylcholine w/w ratio in said micelle is
1:1.33 provides best results. Acceptable results are also obtained for micelles where
sodium deoxycholate to polyvinylpyrrolidone K 30 w/w ratio in said micelle is 1:1,
and sodium deoxycholate to L-α-lysophosphatidylcholine w/w ratio in said micelle is
10 1:1.2.
[0098] These results can be applied to further devise formulations, which exhibit enhanced bioavailability, and can be more effective in delivering appropriate dose of an active molecule to the site of action.
15
36

I/We Claim:
1. A method of preparing micelles or clusters thereof encapsulating fisetin, said method comprising:
(a) contacting fisetin, L-α-lysophosphatidylcholine, at least one surfactant, and at
5 least one water soluble polymer in the presence of at least one organic solvent to
obtain a mixture;
(b) sonicating said mixture to obtain a clear and transparent solution;
(c) evaporating said at least one organic solvent from said solution at a temperature in the range of 40-60°C to obtain a film of micelles or clusters thereof
10 encapsulating fisetin; and
(d) contacting said film with water to obtain a solution of micelles or clusters
thereof encapsulating fisetin.
2. The method as claimed in claim 1, wherein said at least one surfactant is selected
from the group consisting of sodium deoxycholate, sodium cholate, and combinations
15 thereof.
3. The method as claimed in claim 1, wherein said at least one surfactant is sodium deoxycholate.
4. The method as claimed in claim 1, wherein said at least one organic solvent is selected from the group consisting of ethanol, chloroform, methanol, tertiary butanol,
20 trimethylamine, diethyl ether, dichloromethane, trichloromethane, cyclohexane, and
combinations thereof.
5. The method as claimed in claim 1, wherein said at least one organic solvent is
ethanol.
6. The method as claimed in claim 1, wherein said at least one water soluble polymer
25 is selected from the group consisting of albumin, hyaluronic acid, cellulose ethers,
guar gum, carrageenan, dextran, chitosan derivatives, pectins, xanthan gum, poly(ethylene glycol) (PEG), polyvinyl pyrrolidone (PVP), polyvinyl pyrrolidone (PVP) 30, polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyacrylamides, N-(2-
37

hydroxypropyl) methacrylamide, divinyl ether-maleic anhydride (DIVEMA),
polyoxazoline, polyphosphates, polyphosphazenes, and combinations thereof.
7. The method as claimed in claim 1, wherein said at least one water soluble polymer
is polyvinyl pyrrolidone (PVP) 30.
5 8. The method as claimed in claim 1, wherein said mixture is sonicated for one cycle
of five seconds at 25% power for 5 cycles.
9. The method as claimed in claim 1, wherein L-α-lysophosphatidylcholine has a
weight percentage in said mixture in the range of 20%-40%; said at least one
surfactant has a weight percentage in said mixture in the range of 25%- 37%; said at
10 least one water soluble polymer has a weight percentage in said mixture in the range
of 25%- 37%, and fisetin has a weight percentage in said mixture in the range of 2%-8%.
10. A composition comprising micelles or clusters thereof encapsulating fisetin,
prepared by the method as claimed in any of the claims 1-9, wherein
15 (a) fisetin is encapsulated in the interior of the micelles; and
(b) the micelles comprising:
i. L-α-lysophosphatidylcholine;
ii. at least one surfactant; and
iii. at least one water soluble polymer.
20 11. The composition as claimed in claim 10, wherein said at least one surfactant to
said at least one water soluble polymer w/w ratio in said micelle is in the range of 1:0.83-1:1.2, and said at least one surfactant to L-α-lysophosphatidylcholine w/w ratio in said micelle is in the range of 0.5:1.5- 1:2.
12. The composition as claimed in claim 10, wherein said at least one surfactant to
25 said at least one water soluble polymer w/w ratio in said micelle is 1:1, and said at
least one surfactant to L-α-lysophosphatidylcholine w/w ratio in said micelle is 1:1.33.
38

13. The composition as claimed in claim 10, wherein said at least one surfactant to
said water soluble polymer w/w ratio in said micelle is 1:1, and said at least one
surfactant to L-α-lysophosphatidylcholine w/w ratio in said micelle is 1:1.2.
14. A method of increasing fisetin bioavailability in cells, said method comprising:
5 (a) obtaining a composition as claimed in claim 10; and
(b) contacting said composition with cells,
wherein said composition increases fisetin bioavailability in cells.
15. The method as claimed in claim 14, wherein said composition is prepared by a
method as claimed in any of the claims 1-9.