FIELD OF INVENTION
[001] The present disclosure relates to the field of anti-inflammatory compounds, disclosing a composition comprising natural inhibitors of inflammatory responses.
BACKGROUND OF INVENTION
[002] Flavonoids comprise a large group of polyphenolic compounds that are most commonly found occurring in plants as a class of plant pigments. They are widely distributed in most plant species and impart a yellow or red/blue pigmentation to flowers which help in attracting pollinators. Flavonoids are widely consumed as a part of our daily diet and have been shown to have numerous health benefits including anti-carcinogenic, anti-inflammatory properties and prevention of cardiovascular diseases among others.
[003] Among the known flavonoids is fisetin (3,3',4',7-tetrahydroxyflavone), which is found in several fruit and vegetables such as strawberry, apple, persimmon, grape, onion, and cucumber (Khan et al.; Antioxid. Redox Signal.; 2012 ;19(2): 151–162). Studies on this compound have reported several beneficial effects of this flavonoid including neurotrophic, anti-carcinogenic, anti-inflammatory, and other health beneficial effects.
[004] A major limitation to evaluating and elucidating the specific properties of this compound is low aqueous solubility. It has many unique properties like low lipophilicity (CLogP 1.24), high topological polar surface area (tPSA) (107Å), high number of hydrogen bond donors (HBD = 5) and poor bioavailability (Chiruta et al., J Med Chem.; 2012 ;55(1):378-89). The tPSA property makes it less absorbable in the intestine because higher polar surface area introduces stronger H-bonding interactions between intestinal cells and drugs (Hou et al., J Chem Inf Comput Sci.; 2004;44(5):1585-600).

[005] Thus, although in vitro studies have demonstrated the therapeutic properties of this compound, its physical properties have limited thorough pharmacokinetic studies to provide confirmation of its specific therapeutic effects.
SUMMARY OF INVENTION
[006] In an aspect of the present disclosure, there is provided a composition comprising (a) fisetin, and (b) piperine, wherein fisetin to piperine w/w ratio in the said composition is in the range of 1:0.05-1:5.0.
[007] In an aspect of the present disclosure, there is provided a method for preparing a composition, said method comprising: (a) obtaining fisetin, (b) obtaining piperine, and (c) contacting fisetin and piperine to obtain a composition, wherein fisetin to piperine w/w ratio in the said composition is in the range of 1:0.05-1:5.0. [008] These and other features, aspects, and advantages of the present subject matter 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
[009] 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.
[0010] Figure 1 shows the effect of the combination of fisetin and piperine on nitric oxide (NO) production in M1 polarized human monocytes, in accordance with an embodiment of the present disclosure.

[0011] Figure 2 shows the effect of the combination of fisetin and piperine on IL-6
production in M1 polarized human monocytes, in accordance with an embodiment of
the present disclosure.
[0012] Figure 3 shows the effect of the combination of fisetin and piperine on IL-8
production in M1 polarized human monocytes, in accordance with an embodiment of
the present disclosure.
[0013] Figure 4 shows the effect of the combination of fisetin and piperine on LPS
induced NFkB p65 ser536 phosphorylation in macrophage cells, in accordance with an
embodiment of the present disclosure.
[0014] Figure 5 shows the cellular uptake of the combination of fisetin and piperine
in CaCo2 cells, in accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0015] 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 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.
Definitions
[0016] 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 known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0017] 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.
[0018] 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 as
“consists of only”.
[0019] 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.
[0020] The term “including” is used to mean “including but not limited to”.
“Including” and “including but not limited to” are used interchangeably.
[0021] Unless defined otherwise, all technical and scientific terms used herein have
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.
[0022] The present disclosure is not to be limited in scope by the specific
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.
[0023] Herbal compositions have several therapeutic benefits, however their
properties are often not apparent as they less soluble and have low absorption. The
present invention discloses a novel composition of fisetin and piperine which
demonstrates a synergistic increase in anti-inflammatory responses and shows superior
uptake in cells.

[0024] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; and (b) piperine, wherein fisetin
to piperine w/w ratio in said composition is in the range of 1:0.05 – 1:5.0.
[0025] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; and (b) piperine, wherein fisetin
to piperine w/w ratio in said composition is in the range of 1:0.1-1:4.0.
[0026] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; and (b) piperine, wherein fisetin
to piperine w/w ratio in said composition is in the range of 1:0.24 – 1:2.48.
[0027] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; and (b) piperine, wherein fisetin
to piperine w/w ratio in said composition is 1:0.99.
[0028] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; and (b) piperine, wherein fisetin
to piperine w/w ratio in said composition is 1: 1.24.
[0029] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; and (b) piperine, wherein fisetin
to piperine w/w ratio in said composition is 1: 1.48.
[0030] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage of
0.3%- 0.5%; and (b) piperine, wherein fisetin to piperine w/w ratio in said composition
is in the range of 1:0.05 – 1:5.0.
[0031] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage of
0.3%- 0.5%; and (b) piperine, wherein fisetin to piperine w/w ratio in said composition
is in the range of 1:0.1-1:4.0.
[0032] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage of

0.3%- 0.5%; and (b) piperine, wherein fisetin to piperine w/w ratio in said composition
is in the range of 1:0.24 – 1:2.48.
[0033] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage of
0.3%- 0.5%; and (b) piperine, wherein fisetin to piperine w/w ratio in said composition
is 1:0.99.
[0034] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage of
0.3%- 0.5%; and (b) piperine, wherein fisetin to piperine w/w ratio in said composition
is 1: 1.24.
[0035] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage of
0.3%- 0.5%; and (b) piperine, wherein fisetin to piperine w/w ratio in said composition
is 1: 1.48.
[0036] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; and (b) piperine having a
weight percentage of 0.4%- 0.7%wherein fisetin to piperine w/w ratio in said
composition is in the range of 1:0.05 – 1:5.0.
[0037] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; and (b) piperine having a
weight percentage of 0.4%- 0.7%, wherein fisetin to piperine w/w ratio in said
composition is in the range of 1:0.1-1:4.0.
[0038] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; and (b) piperine having a
weight percentage of 0.4%- 0.7%, wherein fisetin to piperine w/w ratio in said
composition is in the range of 1:0.24 – 1:2.48.
[0039] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; and (b) piperine having a

weight percentage of 0.4%- 0.7%, wherein fisetin to piperine w/w ratio in said
composition is 1:0.99.
[0040] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; and (b) piperine having a
weight percentage of 0.4%- 0.7%, wherein fisetin to piperine w/w ratio in said
composition is 1: 1.24.
[0041] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; and (b) piperine having a
weight percentage of 0.4%- 0.7%, wherein fisetin to piperine w/w ratio in said
composition is 1: 1.48.
[0042] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage of
0.3%- 0.5%; and (b) piperine having a weight percentage of 0.4%- 0.7%, wherein
fisetin to piperine w/w ratio in said composition is in the range of 1:0.24 – 1:2.48
[0043] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage of
0.3%- 0.5%; and (b) piperine having weight percentage of 0.4%- 0.7%, wherein fisetin
to piperine w/w ratio in said composition is 1:0.99
[0044] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage of
0.3%- 0.5%; and (b) piperine having weight percentage of 0.4%- 0.7%, wherein fisetin
to piperine w/w ratio in said composition is 1: 1.24.
[0045] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage of
0.3%- 0.5%; and (b) piperine having weight percentage of 0.4%- 0.7%, wherein fisetin
to piperine w/w ratio in said composition is 1: 1.48.
[0046] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; (b) piperine; and (c) suitable

additives and excipients, wherein fisetin to piperine w/w ratio in said composition is in
the range of 1:0.05 – 1:5.0.
[0047] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; (b) piperine; and (c) suitable
additives and excipients, wherein fisetin to piperine w/w ratio in said composition is in
the range of 1:0.1-1:4.0.
[0048] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; (b) piperine; and (c) suitable
additives and excipients, wherein fisetin to piperine w/w ratio in said composition is in
the range of 1:0.24 – 1:2.48.
[0049] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; (b) piperine; and (c) suitable
additives and excipients, wherein fisetin to piperine w/w ratio in said composition is
1:0.99.
[0050] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; (b) piperine; and (c) suitable
additives and excipients, wherein fisetin to piperine w/w ratio in said composition is 1:
1.24.
[0051] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; (b) piperine; and (c) suitable
additives and excipients, wherein fisetin to piperine w/w ratio in said composition is 1:
1.48.
[0052] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage in
the range 0.3%- 0.5%; (b) piperine; and (c) suitable additives and excipients, wherein
fisetin to piperine w/w ratio in said composition is in the range of 1:0.05 – 1:5.0.
[0053] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage in

the range of 0.3%- 0.5%; (b) piperine; and (c) suitable additives and excipients,
wherein fisetin to piperine w/w ratio in said composition is in the range of 1:0.1-1:4.0.
[0054] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage in
the range of 0.3%- 0.5%; (b) piperine; and (c) suitable additives and excipients,
wherein fisetin to piperine w/w ratio in said composition is in the range of 1:0.24 –
1:2.48.
[0055] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage of
0.3%- 0.5%; (b) piperine; and (c) suitable additives and excipients, wherein fisetin to
piperine w/w ratio in said composition is 1:0.99.
[0056] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage of
0.3%- 0.5%; (b) piperine; and (c) suitable additives and excipients, wherein fisetin to
piperine w/w ratio in said composition is 1: 1.24.
[0057] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage of
0.3%- 0.5%; (b) piperine; and (c) suitable additives and excipients wherein fisetin to
piperine w/w ratio in said composition is 1: 1.48.
[0058] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; (b) piperine having a weight
percentage of 0.4%- 0.7%; and (c) suitable additives and excipients, wherein fisetin to
piperine w/w ratio in said composition is in the range of 1:0.05 – 1:5.0.
[0059] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; (b) piperine having a weight
percentage of 0.4%- 0.7%; and (c) suitable additives and excipients, wherein fisetin to
piperine w/w ratio in said composition is in the range of 1:0.1-1:4.0.

[0060] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; (b) piperine having a weight
percentage of 0.4%- 0.7%; and (c) suitable additives and excipients, wherein fisetin to
piperine w/w ratio in said composition is in the range of 1:0.24 – 1:2.48.
[0061] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; (b) piperine having a weight
percentage of 0.4%- 0.7%; and (c) suitable additives and excipients, wherein fisetin to
piperine w/w ratio in said composition is 1:0.99.
[0062] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; (b) piperine having a weight
percentage of 0.4%- 0.7%; and (c) suitable additives and excipients, wherein fisetin to
piperine w/w ratio in said composition is 1: 1.24.
[0063] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin; (b) piperine having a weight
percentage of 0.4%- 0.7%; and (c) suitable additives and excipients, wherein fisetin to
piperine w/w ratio in said composition is 1: 1.48.
[0064] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage of
0.3%- 0.5%; (b) piperine having a weight percentage of 0.4%- 0.7%; and (c) suitable
additives and excipients, wherein fisetin to piperine w/w ratio in said composition is in
the range of 1:0.24 – 1:2.48
[0065] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage of
0.3%- 0.5%; (b) piperine having weight percentage of 0.4%- 0.7%; and (c) suitable
additives and excipients, wherein fisetin to piperine w/w ratio in said composition is
1:0.99.
[0066] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage of

0.3%- 0.5%; (b) piperine having weight percentage of 0.4%- 0.7%; and (c) suitable
additives and excipients, wherein fisetin to piperine w/w ratio in said composition is 1:
1.24.
[0067] In an embodiment of the present disclosure, there is provided a composition to
inhibit inflammatory responses comprising: (a) fisetin having a weight percentage of
0.3%- 0.5%; (b) piperine having weight percentage of 0.4%- 0.7%; and (c) suitable
additives and excipients, wherein fisetin to piperine w/w ratio in said composition is 1:
1.48.
[0068] In an embodiment of the present disclosure, there is provided a method of
preparing a composition comprising: (a) fisetin, and (b) piperine, said method
comprising: (a) obtaining piperine; (b) obtaining fisetin; and (c) contacting piperine
and fisetin to obtain said composition, wherein fisetin to piperine w/w ratio in said
composition is in the range of 1:0.05 – 1:5.0.
[0069] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein comprising: (a) obtaining piperine; (b)
obtaining fisetin; and (c) contacting piperine and fisetin to obtain said composition,
wherein fisetin to piperine w/w ratio in said composition is in the range of 1:0.1-1:4.0.
[0070] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein comprising: (a) obtaining piperine; (b)
obtaining fisetin; and (c) contacting piperine and fisetin to obtain said composition,
wherein fisetin to piperine w/w ratio in said composition is in the range of 1:0.24 –
1:2.48.
[0071] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein comprising: (a) obtaining piperine; (b)
obtaining fisetin; and (c) contacting piperine and fisetin to obtain said composition,
wherein fisetin to piperine w/w ratio in said composition is 1:0.99.
[0072] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein comprising: (a) obtaining piperine; (b)

obtaining fisetin; and (c) contacting piperine and fisetin to obtain said composition,
wherein fisetin to piperine w/w ratio in said composition is 1: 1.24.
[0073] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein comprising: (a) obtaining piperine; (b)
obtaining fisetin; and (c) contacting piperine and fisetin to obtain said composition,
wherein fisetin to piperine w/w ratio in said composition is 1: 1.48.
[0074] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine, wherein fisetin to
piperine w/w ratio in said composition is in the range of 1:0.05 – 1:5.0.
[0075] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine, wherein fisetin to
piperine w/w ratio in said composition is in the range of 1:0.1-1:4.0.
[0076] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine, wherein fisetin to
piperine w/w ratio in said composition is in the range of 1:0.24 – 1:2.48.
[0077] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine, wherein fisetin to
piperine w/w ratio in said composition is 1:0.99.
[0078] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine, wherein fisetin to
piperine w/w ratio in said composition is 1: 1.24.
[0079] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)

fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine, wherein fisetin to
piperine w/w ratio in said composition is 1: 1.48.
[0080] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin; and (b) piperine having a weight percentage of 0.4%- 0.7%wherein fisetin to
piperine w/w ratio in said composition is in the range of 1:0.05 – 1:5.0.
[0081] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin; and (b) piperine having a weight percentage of 0.4%- 0.7%, wherein fisetin to
piperine w/w ratio in said composition is in the range of 1:0.1-1:4.0.
[0082] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin; and (b) piperine having a weight percentage of 0.4%- 0.7%, wherein fisetin to
piperine w/w ratio in said composition is in the range of 1:0.24 – 1:2.48.
[0083] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin; and (b) piperine having a weight percentage of 0.4%- 0.7%, wherein fisetin to
piperine w/w ratio in said composition is 1:0.99.
[0084] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin; and (b) piperine having a weight percentage of 0.4%- 0.7%, wherein fisetin to
piperine w/w ratio in said composition is 1: 1.24.
[0085] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin; and (b) piperine having a weight percentage of 0.4%- 0.7%, wherein fisetin to
piperine w/w ratio in said composition is 1: 1.48.
[0086] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)

fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine having a weight
percentage of 0.4%- 0.7%, wherein fisetin to piperine w/w ratio in said composition is
in the range of 1:0.24 – 1:2.48.
[0087] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine having weight
percentage of 0.4%- 0.7%, wherein fisetin to piperine w/w ratio in said composition is
1:0.99
[0088] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine having weight
percentage of 0.4%- 0.7%, wherein fisetin to piperine w/w ratio in said composition is
1: 1.24.
[0089] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine having weight
percentage of 0.4%- 0.7%, wherein fisetin to piperine w/w ratio in said composition is
1: 1.48.
[0090] In an embodiment of the present disclosure, there is provided a method of
preparing a composition comprising: (a) fisetin, (b) piperine; and (c) suitable additives
and excipients, said method comprising: (a) obtaining piperine; (b) obtaining fisetin;
(c) obtaining suitable additives and excipients; and (d) contacting piperine, fisetin and
suitable additives and excipients to obtain said composition.
[0091] In an embodiment of the present disclosure, there is provided a method of
preparing a composition comprising: (a) fisetin, (b) piperine; and (c) suitable additives
and excipients, said method comprising: (a) obtaining piperine; (b) obtaining fisetin;
(c) obtaining suitable additives and excipients; and (d) contacting piperine, fisetin and

suitable additives and excipients to obtain said composition, wherein fisetin to piperine w/w ratio in said composition is in the range of 1:0.05 – 1:5.0.
[0092] In an embodiment of the present disclosure, there is provided a method of preparing a composition as described herein, wherein said method comprises: (a) obtaining piperine; (b) obtaining fisetin; (c) obtaining suitable additives and excipients; and (d) contacting piperine, fisetin and suitable additives and excipients to obtain said composition, wherein fisetin to piperine w/w ratio in said composition is in the range of 1:0.1-1:4.0.
[0093] In an embodiment of the present disclosure, there is provided a method of preparing a composition as described herein, wherein said method comprises: (a) obtaining piperine; (b) obtaining fisetin; (c) obtaining suitable additives and excipients; and (d) contacting piperine, fisetin and suitable additives and excipients to obtain said composition, wherein fisetin to piperine w/w ratio in said composition is in the range of 1:0.24 – 1:2.48.
[0094] In an embodiment of the present disclosure, there is provided a method of preparing a composition as described herein, wherein said method comprises: (a) obtaining piperine; (b) obtaining fisetin; (c) obtaining suitable additives and excipients; and (d) contacting piperine, fisetin and suitable additives and excipients to obtain said composition, wherein fisetin to piperine w/w ratio in said composition is 1:0.99. [0095] In an embodiment of the present disclosure, there is provided a method of preparing a composition as described herein, wherein said method comprises: (a) obtaining piperine; (b) obtaining fisetin; (c) obtaining suitable additives and excipients; and (d) contacting piperine, fisetin and suitable additives and excipients to obtain said composition, wherein fisetin to piperine w/w ratio in said composition is 1: 1.24. [0096] In an embodiment of the present disclosure, there is provided a method of preparing a composition as described herein, wherein said method comprises: (a) obtaining piperine; (b) obtaining fisetin; (c) obtaining suitable additives and excipients;

and (d) contacting piperine, fisetin and suitable additives and excipients to obtain said
composition, wherein fisetin to piperine w/w ratio in said composition is 1: 1.48.
[0097] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine, wherein fisetin to
piperine w/w ratio in said composition is in the range of 1:0.05 – 1:5.0.
[0098] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine, wherein fisetin to
piperine w/w ratio in said composition is in the range of 1:0.1-1:4.0.
[0099] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine, wherein fisetin to
piperine w/w ratio in said composition is in the range of 1:0.24 – 1:2.48.
[00100] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine, wherein fisetin to
piperine w/w ratio in said composition is 1:0.99.
[00101] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine, wherein fisetin to
piperine w/w ratio in said composition is 1: 1.24.
[00102] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine, wherein fisetin to
piperine w/w ratio in said composition is 1: 1.48.
[00103] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)

fisetin; and (b) piperine having a weight percentage of 0.4%- 0.7%wherein fisetin to
piperine w/w ratio in said composition is in the range of 1:0.05 – 1:5.0.
[00104] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin; and (b) piperine having a weight percentage of 0.4%- 0.7%, wherein fisetin to
piperine w/w ratio in said composition is in the range of 1:0.1-1:4.0.
[00105] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin; and (b) piperine having a weight percentage of 0.4%- 0.7%, wherein fisetin to
piperine w/w ratio in said composition is in the range of 1:0.24 – 1:2.48.
[00106] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin; and (b) piperine having a weight percentage of 0.4%- 0.7%, wherein fisetin to
piperine w/w ratio in said composition is 1:0.99.
[00107] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin; and (b) piperine having a weight percentage of 0.4%- 0.7%, wherein fisetin to
piperine w/w ratio in said composition is 1: 1.24.
[00108] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin; and (b) piperine having a weight percentage of 0.4%- 0.7%, wherein fisetin to
piperine w/w ratio in said composition is 1: 1.48.
[00109] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine having a weight
percentage of 0.4%- 0.7%, wherein fisetin to piperine w/w ratio in said composition is
in the range of 1:0.24 – 1:2.48

[00110] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine having weight
percentage of 0.4%- 0.7%, wherein fisetin to piperine w/w ratio in said composition is
1:0.99
[00111] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine having weight
percentage of 0.4%- 0.7%, wherein fisetin to piperine w/w ratio in said composition is
1: 1.24.
[00112] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein said composition comprises: (a)
fisetin having a weight percentage of 0.3%- 0.5%; and (b) piperine having weight
percentage of 0.4%- 0.7%, wherein fisetin to piperine w/w ratio in said composition is
1: 1.48.
[00113] In an embodiment of the present disclosure, there is provided a composition
comprising (a) fisetin; and (b) piperine, wherein said composition enhances cellular
uptake of fisetin, when administered to cells for a period in the range of 4-8 hours.
[00114] In an embodiment of the present disclosure, there is provided a composition
comprising (a) fisetin; and (b) piperine, wherein said composition enhances cellular
uptake of fisetin, when administered to cells for a period of 6 hours.
[00115] In an embodiment of the present disclosure, there is provided a composition
comprising (a) fisetin; and (b) piperine, in the w/w ratio of fisetin: piperine in the range
of 1:0.05 – 1:5.0, wherein said composition enhances cellular uptake of fisetin, when
administered to cells for a period of 4-8 hours.
[00116] In an embodiment of the present disclosure, there is provided a composition
comprising (a) fisetin; and (b) piperine, in the w/w ratio of fisetin: piperine in the range

of 1:0.05 – 1:5.0, wherein said composition enhances cellular uptake of fisetin, when
administered to cells for a period of 6 hours.
[00117] In an embodiment of the present disclosure, there is provided a composition
comprising (a) fisetin; and (b) piperine, in the w/w ratio of fisetin: piperine in the range
of 1:0.1-1:4.0, wherein said composition enhances cellular uptake of fisetin, when
administered to cells for a period of 6 hours.
[00118] In an embodiment of the present disclosure, there is provided a composition
comprising (a) fisetin; and (b) piperine, in the w/w ratio of fisetin: piperine in the range
of 1:0.24 – 1:2.48, wherein said composition enhances cellular uptake of fisetin, when
administered to cells for a period of 6 hours.
[00119] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) fisetin; (b) piperine; and (c) suitable additives and excipients, wherein
said composition is a liquid.
[00120] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) fisetin; (b) piperine; and (c) suitable additives and excipients.
[00121] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) fisetin; (b) piperine; and (c) suitable additives and excipients, wherein
said composition is a spray.
[00122] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein said carrier can be substances which serve as mechanisms
to improve the delivery and the effectiveness of active ingredients.
[00123] 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 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
[00124] 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 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 methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.
Example 1
Materials and methods
[00125] 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)] 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 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), piperine (P49007), fisetin F4043) and phorbol 12-myristate 13-acetate (P8139) and Griess’ reagent for nitrite (03553) were purchased from Sigma-Aldrich.

[00126] NFKB p65 (RelA) ser536 assay: RAW macrophage cells were seeded into each well of 12-well plates until 80% confluent. NFKB p65 ser 536 phosphorylation was stimulated with (10 pg/ml) lipopolysaccharides (LPS). This treatment induces the inflammatory response within 90 minutes of stimulation. Simultaneously, the cells were treated with fisetin and piperine. Detection of NFKB 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 (PBS) (1X). Cells were lysed with 0.5 mL ice cold 1X cell lysis buffer (20 mM Tris-HCl (pH 7.5); 150 mM NaCl; 1mM Na2EDTA; 1mM EGTA; 1% Triton; 2.5 mM sodium pyrophosphate; 1 mM beta-glycerophosphate; 1 mM Na3VO4; 1 ug/mL leupeptin; 1 mM 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 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 revolutions per minute (RPM) for 10 minutes at 4 ºC and transferred to a new tube. Cell lysates, when needed, were diluted with sample diluents supplied with the kit {Cell signaling Inc (cat # 7173C)}. The lysates were then transferred to 96 well plates and incubated overnight at 37 ºC. Next, washing of the wells was done with 1X wash buffer (supplied with kit). To each well, 100 [xL of NF-KB p65 detection antibody (supplied with kit) was added and the plate was incubated at 37 ºC for one-hour. The wells were then washed again and 100 uL of horseradish peroxidase (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 |iL of 3, 3', 5, 5'-tetramethylbenzidine (TMB) buffer was added. Finally, the stop solution was added and absorbance read at 450 nm using a Multiskan Go microplate reader (Thermo Fisher, Germany).
[00127] 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 morphology owing to their attachment to the substrate. The cells treated with 25 mg/ml of LPS for the last 12 h of PMA. To confirm the macrophage polarization, the amount 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 manufacturer’s instructions. [00128] Nitric Oxide Production: Polarized M1 cells were treated with co-administration of piperine and 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 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. [00129] IL-6 and IL-8 cytokine production: Polarized M1 cells were treated with co-administration of piperine and 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). [00130] 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 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 fisetin and co-administration of piperine for 6 hours. 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.
Example 2:
Effect of the combination of piperine and fisetin on nitric oxide production in M1
polarized human monocytes
[00131] The combination of piperine and fisetin has never been tested identify a role in the inhibition of inflammatory responses. Table 1 depicts the various combinations of piperine: fisetin used in the disclosed invention.

[00132] In order to determine if the composition of fisetin and piperine would have enhanced efficacy in inhibiting inflammatory responses, the different combinations were tested on M1 polarized monocytes. Monocytes undergo polarization to form the M1 subtype of macrophages, in response to environmental stressors such as cellular damage and microbial infection among others. Following polarization, they exhibit a suite of cellular responses, including production of pro-inflammatory cytokines and production of NO and reactive oxygen species. Therefore, NO production by M1 macrophages was tested following treatment of macrophage cells with varying concentrations of fisetin and piperine. As shown in Figure 1, curcumin, a well-known anti-inflammatory agent, inhibits NO production by 63.3%. Fisetin and Piperine, in isolation, exhibit slightly reduced capacity to inhibit NO production, at 47.3 and 43.2% respectively. Surprisingly, combinations of fisetin and piperine prove to be even better than curcumin in reducing NO production. The ratio of fisetin and piperine at 1:1.48 (Table 1) shows 72% decrease in NO production. The other ratios also show a good reduction in production of NO ranging from 57.6% to 69.3%.
[00133] Thus, as compared to the individual components, the ratios 1: 0.99; 1: 1.24 and 1: 1.48, remarkably, show a specific synergistic effect and decrease the production of NO in polarized macrophages by 69.3%, 69.1% and 72%respectively.

Example 3
Effect of the combination of piperine and fisetin on IL-6 and IL-6 production in
M1 polarized human monocytes
[00134] To test the efficacy of the compositions further, the effect of fisetin and piperine in the reduction of pro-inflammatory molecules was tested. Interleukin-6 (IL-6) and interleukin-8 (IL-8) are chemokines also produced in the defence responses of M1 polarized macrophages. IL-6 has two receptors, namely membrane-bound β-receptor glycoprotein 130 (gp130) and soluble forms of the IL-6R (sIL-6R) (Wolf et al., Cytokine.; 2014; 70(1):11-20). IL-8 functions as a regulator of leukocyte recruitment and trafficking. It has two receptors namely CXCR1 and CXCR2 (Huang et al., Am J Pathol.; 2005; 166(6):1807-15). The receptors of inflammatory mediators like IL-6 and IL-8 could potentially be influenced by the combination of fisetin and piperine. The results demonstrate that, similar to the inhibition of NO production, fisetin and piperine at ratios of 1: 0.99; 1: 1.24 and 1: 1.48 show synergism in the inhibition of pro-inflammatory markers (Figures 2 and 3). Fisetin in combination with piperine, at the aforesaid ratios shows inhibition in IL-6 production by 54.5%, 54.0% and 55.2% respectively (Figure 2). Similarly, the same ratios of fisetin: piperine at 1:0.99; 1:1.24 and 1:1.48 also show 73.7%, 73.5% and 76.0% inhibition in IL-8 production when compared with fisetin alone treatment (Figure 3).
Example 4
Effect of piperine and fisetin on LPS induced NFkB p65 ser536 phosphorylation
in macrophages
[00135] The NFkB group of proteins are expressed in M1 macrophages as a common response to microbial infection. They are produced in response to bacterial antigens and surface molecules, such as lipopolysaccharides (LPS). To ascertain whether the composition of fisetin and piperine was effective against anti-inflammatory responses

elicited by microbial infection, NFkB p65 ser536 phosphorylation in macrophages was
tested.
[00136] The combination at the synergistic w/w ratio of 1:1.48 of fisetin: piperine (see
Table 1) was tested. Figure 4 demonstrates that the combination of fisetin and piperine
at the ratio 1:1.48, shows better anti-inflammatory response (inhibition of NFkB p65
ser536 phosphorylation) (14.3% and 31.24%, respectively) when compared with fisetin
and piperine alone respectively.
Example 5
Combination of fisetin and piperine with enhanced bio-availability
[00137] Fisetin is a naturally occurring flavonoid and has anticancer and
antiangiogenic properties. However, a severe limitation to its use is the low solubility
and absorption of this compound. (Seguin et al., Int J Pharm.; 2013; 444(1-2):146-54).
In order to overcome this problem, piperine was co-administered with fisetin. Piperine
acts to increase the absorption of compounds by increasing blood supply to the
gastrointestinal tract (Feng et al., Molecules.; 2014; 19(5):5624-33).
[00138] Piperine concentrations ranging from 0.71 μg/ml to 7.13 μg/ml (Table 1) was
co-administered with fisetin in Caco-2 cells, for a period of 6 hours. As seen from
Figure 5, increasing concentrations of piperine cause an increase in absorption of the
composition. The co-administration of piperine from 10% to 50% in the composition,
increases the absorption of the combination of fisetin and piperine in a dose dependent
manner.
[00139] Thus, the cellular absorption of fisetin shows a significant enhancement when
delivered in combination with piperine.
[00140] Overall, the above examples illustrate that the combination of fisetin and
piperine in specific ratios, shows remarkable synergy in inhibiting anti-inflammatory
responses, in comparison to the compounds taken in isolation. The enhanced capacity
of the composition of fisetin and piperine, to inhibit anti-inflammatory responses

triggered by varied environmental triggers, is particularly efficacious within the ranges of 1: 0.99; 1: 1.24 and 1: 1.48 of fisetin: piperine.

I/We Claim:
1. A composition comprising:
(a) fisetin; and
(b) piperine,
wherein fisetin to piperine w/w ratio in said composition is in the range of 1:0.05 – 1: 5.0
2. The composition as claimed in claim 1, wherein fisetin to piperine w/w ratio in said composition is in the range of 1:0.1- 1:4.0.
3. The composition as claimed in claim 1, wherein fisetin to piperine w/w ratio in said composition is in the range of 1:0.24 – 1:2.48.
4. The composition as claimed in any of the claims 1-3, further comprising suitable additives and excipients.
5. The composition as claimed in any of the claims 1-4, wherein said composition is effective in inhibition of inflammatory responses.
6. A method for preparing a composition as claimed in claim 1, said method
comprising:
(a) obtaining fisetin;
(b) obtaining piperine; and
(c) contacting fisetin and piperine to obtain a composition.
7. A method for preparing a composition as claimed in claim 4, said method
comprising:
(a) obtaining fisetin;
(b) obtaining piperine;
(c) obtaining suitable additives and excipients; and
(d) contacting fisetin, piperine, and suitable additives and excipients to obtain a
composition.

8. The composition as claimed in any of the claims 1-7, wherein said composition has superior cellular uptake when administered to cells for a period in the range of 4-8 hours.