TECHNICAL FIELD
[0001] The present invention relates to nanoparticles. Specifically, the present invention
relates to nanoparticles of Imperatorinhaving anti-cancer activity. The present invention further relates to biocompatible and biodegradable PLGA linked nanoparticles of Imperatorin, exhibiting enhanced anti-cancer activity and their method of preparation.
BACKGROUND
[0002] Background description includes information that may be useful in understanding the
10 present invention. It is not an admission that any of the information provided herein is prior art
or relevant to the presently claimed invention, or that any publication specifically or implicitly
referenced is prior art.
[0003] Imperatorin is a furocoumarin and a phytochemical that has been isolated from
Urenalobata L. (Malvaceae), Angelica archangelica, Angelica dahurica, Glehnialittoralis,
15 Saposhnikoviadivaricata, Cnidiummonnieri, Incarvillea younghusbandii,
andZanthoxylumamericanum mill. It is biosynthesized from umbelliferone, a coumarin
derivative.
20 [0004] Physically, the pure Imperatorinis present as white, long fine needles or crystals with
melting point of 101 ᵒC. Imperatorin is insoluble in water and is easily soluble in nonpolar
solvents (Cox et al. 2003). Many of the pharmacological activities observed in ethnomedicine
Imperatorin became the subject of profound studies, and a series of important biological
properties were described which indicated that imperatorin is an important bioactive molecule.
- 3 -
[0005] Activity of imperatorin on the central nervous system (CNS): The influence of
imperatorin on processes of learning, its anxiolytic effect and anti-epileptic activity were have
been studied extensively.
[0006] Influence on the cardio-vascular system: Imperatorin was examined as both an
antihypertensive and as a cardioprotecting agent. Imperatorin showed a significant reduction 5 on in
systolic and diastolic blood pressure in mice. The vascular smooth muscle seems to be the target
tissue for imperatorin, and probably has the same mechanism of action as typical calcium
antagonists like nitrendypine and verapamil, although it is more similar to the phenylalkylamines
(Zhang et al. 2011b). The effects of different extracts of the aerial parts of Angelica dahurica var.
10 framosana on the vascular relaxation of phenylephrine-induced mouse thoracic aorta contraction
were investigated. The most active cyclohexane and ethyl acetate extracts were found to have the
highest concentrations of imperatorin (4.09 and 1.70 %, respectively) and showed IC50 values of
35.3 and 40.5 mg l-1, respectively. The effect of imperatorin itself was dose dependent.
Imperatorin, compared to isoimperatorin had a four-fold stronger vasodilatation effect (IC50 =
15 12.2 and 47.6lmol l-1). The vasodilatation effect of imperatorin was significantly attenuated to
24.88 % in the denuded endothelium group compared with the intact endothelium group (Nie et
al. 2009).
[0007] Antibacterial activity: Furanocoumarins, which are known to possess broad
antimicrobial properties, merit further attention to explore their range of activity and potential as
20 lead structures for further development (Stavri and Gibbons 2005). Plants containing imperatorin
have a strong justification in bacterial infections in traditional medicine. Imperatorin, together
with other coumarins, such as isoimperatorin, phellopterin, byakangelicin, and scopoletin were
tested against Bacillus subtilis, Escherichia coli, Cladosporiumherbarum, and Aspergillus
candidus. In most cases, imperatorin was inactive with an MIC of 1000 lg ml-1, and weak
25 antimicrobial activity was observed against B. subtilis (MIC = 500 lg ml-1) (Kwon et al. 2002).
Similar results were obtained by Kwon et al. (Kwon et al. 1997). In experiments conducted by
Sarker et al. (2007), imperatorin was active against Gram-positive bacteria like Bacillus cereus
(MIC = 5 9 10-5 lg ml-1).
[0008] Antiviral activity: influence on replication HIV:Imperatorin was found to be an HIV-
30 1 replication inhibitor. The activity of imperatorin was examined on several cell lines: human
- 4 -
cervical carcinoma cell (HeLa), human T leukemia cells (MT-2) and Jurkat T cells. Imperatorin
strongly inhibits cyclin D1 expression and arrests the cells at the G1 phase of the cell cycle. HIV-
1 replication follows a Sp1- dependent pathway and is directly inhibited by this furanocoumarin
(Sancho et al. 2004).
[0009] Anticancer activity of imperatorin: Many papers report the anticancer effects 5 ts of
imperatorin and few mechanisms of this activity were reported, including anoikis, autophagy
induction, necrosis, and inhibition of the formation of DNA adducts. The influence of
imperatorin on HepG2, SPCA1, SGC-7901, Bcap-37, MCF-7, HeLa, HT29, HL- 60, and K562
cell lines was studied and Imperatorin significantly reduces HeLa cell and laryngeal carcinoma
10 (Hep-2) cell viability by induction of apoptosis, increasing the activity mediators of apoptosiscaspase-
3 and caspase-8 for both cell lines. Imperatorin also induces the activities of carcinogendetoxifying
enzymes, such as glutathione S-transferase (GST) and oxidoreductase (NQO1).
[0010] Other pharmacological properties: Imperatorin was examined as a bone loss inhibitor.
It improved osteo-induction by increasing BMP-2 gene expression more than threefold at a dose
15 of 10 ml/mol compared with control. Collagen content in the tissue and mineralization were also
increased with a higher concentration of imperatorin (Chen 2011).
[0011] Imperatorin also demonstrates hepatoprotective action. Imperatorin was found to
inhibit the elevation of plasma alanine aminotransferase (ALT) in mice with concanavalin Ainduced
hepatitis and mice anti-Fas antibody-induced hepatitis. Imperatorin was also examined
20 for its effects on macrophage functions of relevance to the inflammatory process. Ionophorestimulated,
mouse peritoneal macrophages served as a source of cyclooxygenase-1 (COX-1) and
5-lipooxygenase (5-LOX).
[0012] Pharmacokinetics of imperatorin: The kinetics of a drug describes its behavior in an
organism in five steps: liberation, absorption, distribution, metabolisation, and excretion. The
25 liberation step is important with respect to the pharmaceutical presentation of the drug. The
absorption process after the oral administration of imperatorin in rats was described as slow and
the bioavailability as poor. After i.e. administration of imperatorin, the extravascular distribution
was low, and the compound was rapidly eliminated.
- 5 -
[0013] A very important issue in determining the pharmacokinetic properties of a drug is
establishing its ability to cross the blood–brain barrier (BBB). As a relatively non-polar
molecule, imperatorin passes easily through the BBB and is a highly permeable compound. Two
mechanisms for this process were considered: transcellular passive diffusion and active
transportation, which involves influx and efflux transporters. The P-gp protein (permeabilit5 y
glycoprotein) is a product of the multidrug resistance (mdr) gene and it is considered that PGP
functions as an efflux system at the BBB.
[0014] Imperatorin has different distribution in various rat brain tissues. The highest
concentrations were detected in the striatum, hippocampus, brain stem, cortex, cerebellum, and
10 diencephalon, respectively. The half-life in the hippocampus was significantly longer than in
other structures (Zhang et al. 2011a).
[0015] The only formulations available for Imperatorin are sustained release tablets. From
the available literature, it is clear that imperatorin is highly soluble in non-polar solvents like
methanol, ethanol, ethylacetate, hexane etc. and shows anticancer activity due to its ability to
15 cross the blood brain barrier (BBB).
[0016] There is,therefore, a need to develop more biocompatible and effective formulations
that can enhance the activity profile of Imperatorin while still retaining its safety profile. The
present invention provides a nanoparticle based formulation of Imperatorin which is more
effective than conventional formulations available.
20
OBJECTS OF THE INVENTION
[0017] An object of the present invention is to provide nanoparticles of Imperatorin which
are biodegradable.
[0018] Yet another object of the present invention is to provide nanoparticles of Imperatorin
25 with toxicologically safe in vivo degradation products.
[0019] Another object of the present invention is to provide nanoparticles of Imperatorin
which can be easily eliminated by the metabolic pathways.
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[0020] An object of the present invention is to provide nanoparticles of Imperatorin which
offer site specific delivery of drugs.
[0021] Another object of the present invention is to provide nanoparticles of Imperatorin,
having enhanced anti-cancer properties.
[0022] Yet another object of the present invention is to provide formulation of nanoparticle5 s
of imperatorin using PLGA, with increased target specification.
[0023] Another object of the present invention is to provide anticancer formulations for safe
herbal materials with enhanced absorption.
[0024] The other objects and preferred embodiments and advantages of the present invention
10 will become more apparent from the following description of the present invention when read in
conjunction with the accompanying examples and figures, which are not intended to limit scope
of the present invention in any manner.
SUMMARY OF THE INVENTION
15 [0025] This summary is provided to introduce a selection of concepts in a simplified form
that are further described below in Detailed Description section. This summary is not intended to
identify key features or essential features of the claimed subject matter, nor is it intended to be
used as an aid in determining the scope of the claimed subject matter.
[0026] The present invention relates to nanoparticles. Specifically, the present invention
20 relates to nanoparticles of Imperatorin having anti-cancer activity.
[0027] In one aspect, the present invention relates to biocompatible and biodegradable PLGA
linked nanoparticles of Imperatorin.
[0028] In another aspect, the present invention relates to PLGA linked nanoparticles of
Imperatorin, exhibiting enhanced anti-cancer activity.
25 [0029] In one aspect, the present invention relates to formulation of nanoparticles of
imperatorin using PLGA, having increased target specification.
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[0030] In yet another aspect, the present invention relates to a method of preparation of
PLGA based nanoparticles of Imperatorin, which demonstrate excellent anti-cancer properties.
[0031] In still another aspect, the present invention relates to anticancer formulations for safe
herbal materials with enhanced absorption.
[0032] In one aspect, the present invention relates to a method of preparation of PLGA base5 d
nanoparticles of Imperatorin, using dispersion of pre-formed polymers by solvent evaporation
method.
[0033] In another aspect, the present invention relates to a method of preparation of
nanoparticles of Imperatorin, comprising the following steps:
10 a) Dissolving Imperatorin in organic solvent and adding to a solution of chosen
surfactant in water;
b) Stirring the resulting mixture to evaporate the organic solvent by air drying;
c) Homogenising the suspension;
d) Sonicating the suspension;
15 e) Centrifugation followed by separation of supernatant;
f) Separating the nanoparticles as sediments and resuspending them in water; and
g) Freeze drying the suspension to obtain nanoparticles of Imperatorin.
[0034] In another aspect, the present invention relates to a method of preparation of PLGA
based nanoparticles of Imperatorin, comprising the following steps:
20 [0035] Other aspects of the invention will be set forth in the description which follows, and
in part will be apparent from the description, or may be learnt by the practice of the invention.
DETAILED DESCRIPTION
[0036] The following is a detailed description of embodiments of the disclosure. The
embodiments are in such detail as to clearly communicate the disclosure. However, the amount
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of detail offered is not intended to limit the anticipated variations of embodiments; on the
contrary, the intention is to cover all modifications, equivalents, and alternatives falling within
the spirit and scope of the present disclosure as defined by the appended claims.
[0037] All publications herein are incorporated by reference to the same extent as if each
individual publication or patent application were specifically and individually indicated to 5 be
incorporated by reference. Where a definition or use of a term in an incorporated reference is
inconsistent or contrary to the definition of that term provided herein, the definition of that term
provided herein applies and the definition of that term in the reference does not apply.
[0038] Reference throughout this specification to “one embodiment” or “an embodiment”
10 means that a particular feature, structure or characteristic described in connection with the
embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one
embodiment” or “in an embodiment” in various places throughout this specification are not
necessarily all referring to the same embodiment. Furthermore, the particular features, structures,
or characteristics may be combined in any suitable manner in one or more embodiments.
15 [0039] In some embodiments, the numbers expressing quantities of ingredients, properties
such as concentration, reaction conditions, and so forth, used to describe and claim certain
embodiments of the invention are to be understood as being modified in some instances by the
term “about. “Accordingly, in some embodiments, the numerical parameters set forth in the
written description and attached claims are approximations that can vary depending upon the
20 desired properties sought to be obtained by a particular embodiment. In some embodiments, the
numerical parameters should be construed in light of the number of reported significant digits
and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and
parameters setting forth the broad scope of some embodiments of the invention are
approximations, the numerical values set forth in the specific examples are reported as precisely
25 as practicable.The numerical values presented in some embodiments of the invention may
contain certain errors necessarily resulting from the standard deviation found in their respective
testing measurements.
[0040] As used in the description herein and throughout the claims that follow, the meaning
of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise.
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Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the
context clearly dictates otherwise.
[0041] Unless the context requires otherwise, throughout the specification which follow, the
word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be
construed in an open, inclusive sense that is as “including, but not limited 5 ted to.”
[0042] The recitation of ranges of values herein is merely intended to serve as a shorthand
method of referring individually to each separate value falling within the range.Unless otherwise
indicated herein, each individual value is incorporated into the specification as if it were
individually recited herein. All methods described herein can be performed in any suitable order
10 unless otherwise indicated herein or otherwise clearly contradicted by context.The use of any and
all examples, or exemplary language (e.g. “such as”) provided with respect to certain
embodiments herein is intended merely to better illuminate the invention and does not pose a
limitation on the scope of the invention otherwise claimed. No language in the specification
should be construed as indicating any non-claimed element essential to the practice of the
15 invention.
[0043] Groupings of alternative elements or embodiments of the invention disclosed herein
are not to be construed as limitations. Each group member can be referred to and claimed
individually or in any combination with other members of the group or other elements found
herein. One or more members of a group can be included in, or deleted from, a group for reasons
20 of convenience and/or patentability. When any such inclusion or deletion occurs, the
specification is herein deemed to contain the group as modified thus fulfilling the written
description of all Markush groups used in the appended claims.
[0044] The description that follows, and the embodiments described therein, is provided by
way of illustration of an example, or examples, of particular embodiments of the principles and
25 aspects of the present disclosure. These examples are provided for the purposes of explanation,
and not of limitation, of those principles and of the disclosure.
[0045] It should also be appreciated that the present disclosure can be implementedin
numerous ways, including as a system, a method or a device. In this specification, these
implementations, or any other form that the invention may take, may be referred to as processes.
- 10 -
In general, the order of the steps of the disclosed processes may be altered within the scope of
the invention.
[0046] The headings and abstract of the invention provided herein are for convenience only
and do not interpret the scope or meaning of the embodiments.
[0047] The following discussion provides many example embodiments of the inventi5 ve
subject matter. Although each embodiment represents a single combination of inventive
elements, the inventive subject matter is considered to include all possible combinations of the
disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second
embodiment comprises elements B and D, then the inventive subject matter is also considered to
10 include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[0048] Various terms as used herein are shown below. To the extent a term used in a claim
is not defined below, it should be given the broadest definition persons in the pertinent art have
given that term as reflected in printed publications and issued patents at the time of filing.
[0049] Imperatorin is a furocoumarin and a phytochemical that has been isolated from
15 Urenalobata L. (Malvaceae), Angelica archangelica, Angelica dahurica, Glehnialittoralis,
Saposhnikoviadivaricata, Cnidiummonnieri, Incarvillea younghusbandii, and
Zanthoxylumamericanum mill. It is biosynthesized from umbelliferone, a coumarin derivative.
[0050] Imperatorin shows a relatively low bioavailability because of its poor water
20 solubility. Thus, it is difficult to prepare an ideal oral pharmaceutical preparation. It is more
difficult to be developed as an injection. Imperatorin has great potential to be developed as a
pharmaceutical formulation for subsequent clinical assessment based on its poor solubility in
water and good fat solubility.
[0051] Nanoparticles represent drug delivery systems suitable for most administration
25 routes. Over the years, a variety of natural and synthetic polymers have been explored for the
- 11 -
preparation of nanoparticles, of which Poly(lactic acid) (PLA), Poly(glycolic acid) (PGA), and
their copolymers (PLGA) have been extensively investigated because of their biocompatibility
and biodegradability. Nanoparticles act as potential carries for several classes of drugs such as
anticancer agents, antihypertensive agents, immunomodulators, and hormones; and
macromolecules such as nucleic acids, proteins, peptides, and anti5 bodies.
[0052] PLGA is a linear copolymer with lactic and glycolic acid repeat units, which can be
organized as a block-co-polymer or statistical polymer. The body can easily metabolize both
monomers into carbon dioxide and water via the tricarboxylic acid cycle, resulting in minimal
systemic toxicity.
10 [0053] The present invention relates to nanoparticles. Specifically, the present invention
relates to nanoparticles of Imperatorinhaving anti-cancer activity.
[0054] In one embodiment, the present invention relates to biocompatible and
biodegradable PLGA linked nanoparticles of Imperatorin.
[0055] In another embodiment, the present invention relates to PLGA linked nanoparticles
15 of Imperatorin, exhibiting enhanced anti-cancer activity.
[0056] The present invention discloses nanoparticles of Imperatorin and their preparation.
The targeting capability of nanoparticles is influenced by particle size, surface charge, surface
modification, and hydrophobicity, whereas the performance of nanoparticles in vivo is
influenced by morphological characteristics, surface chemistry, and molecular weight. To
20 prepare nanoparticles with controlled properties, such as size or degradation rate, it is important
to choose not only the right method, but also the right materials and synthesis parameters. For
example, particle size can be affected by the amount of surfactant, polymer concentration,
encapsulated compound, and the type and extent of energy input (e.g. sonication,
homogenization or shear stirring).
25 [0057] Poly (lactic-co-glycolic acid) (PLGA) is one of the most successfully developed
biodegradable polymers. Among the different polymers developed to formulate polymeric
nanoparticles, PLGA has attracted considerable attention due to its attractive properties: (i)
biodegradability and biocompatibility, (ii) FDA and European Medicine Agency approval in
- 12 -
drug delivery systems for parenteral administration, (iii) well described formulations and
methods of production adapted to various types of drugs e.g.hydrophilic or hydrophobic small
molecules or macromolecules, (iv) protection of drug from degradation, (v) possibility of
sustained release, (vi) possibility to modify surface properties to provide stealthness and/or better
interaction with biological materials and (vii) possibility to target nanoparticles to specific orga5 ns
or cells. Therefore, PLGA has been chosen to design nanoparticles as drug delivery systems in
cancer.
[0058] In one embodiment, the present invention relates to formulation of nanoparticles of
imperatorin using PLGA, having increased target specification.
10 [0059] In yet another embodiment, the present invention relates to a method of preparation
of PLGA based nanoparticles of Imperatorin, which demonstrate excellent anti-cancer properties.
[0060] Dispersion of pre-formed polymers is the most common technique used to
prepare biodegradable nano-particles from PLA, PLG, PLGA, PCA etc.
[0061] Solvent evaporation method: Solvent evaporation is usually carried out by
15 continuous stirring at ambient pressure, but its rate can be accelerated under reduced pressure,
vacuum, or elevated temperature. DCM, which has a low boiling point of 39.8°C and negligible
water solubility (1.32 wt%), is the preferred solvent of choice. This technique is particularly
useful for loading hydrophobic drugs into PLGA nanoparticles. Prior to emulsification, an
emulsifier is added to a continuous phase and/or a PLGA organic solution. Common examples of
20 surfactants and/or stabilizers are polyvinyl alcohol (PVA), didodecyldimethylammonium
bromide, polyvinyl pyrrolidone, solutol, polysorbate, poloxamer, carbopol, polyethylene glycol
(PEG), sodium dodecyl sulfate, proteins, carbohydrates, lecithin, and PEG-lipid (e.g., PEGceramide,
d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS)).
[0062] In still another embodiment, the present invention relates toanticancer formulations
25 for safe herbal materials with enhanced absorption.
[0063] In one embodiment, the present invention relates to a method of preparation of
PLGA based nanoparticles of Imperatorin, using dispersion of pre-formed polymers by solvent
evaporation method.
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[0064] In another embodiment, the present invention relates to a method of preparation of
nanoparticles of Imperatorin, comprising the following steps:
h) Dissolving Imperatorin in organic solvent and adding to a solution of chosen
surfactant in water;
i) Stirring the resulting mixture to evaporate the organic solvent by air drying5 ;
j) Homogenising the suspension;
k) Sonicating the suspension;
l) Centrifugation followed by separation of supernatant;
m) Separating the nanoparticles as sediments and resuspending them in water; and
10 n) Freeze drying the suspension to obtain nanoparticles of Imperatorin.
[0065] In another embodiment, the present invention relates to a method of preparation of
PLGA based nanoparticles of Imperatorin, comprising the following steps:
[0066] In another embodiment, the present invention relates to evaluation of the
synthesized nanoparticles using zeta potential, Transmission electron microscopy, Differential
15 scanning calorimetry, X-ray powder diffractometry and High-performance liquid
chromatography, to measure the drug loading, encapsulation efficiency, and in-vitro drug
release.
[0067] While the foregoing describes various embodiments of the disclosure, other and
further embodiments of the disclosure may be devised without departing from the basic scope
20 thereof. The scope of the invention is determined by the claims that follow. The invention is not
limited to the described embodiments, versions or examples, which are included to enable a
person having ordinary skill in the art to make and use the invention when combined with
information and knowledge available to the person having ordinary skill in the art.
EXAMPLES
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[0068] The present invention is further explained in the form of following examples.
However, it is to be understood that the following examples are merely illustrative and are not to
be taken as limitations upon the scope of the invention.
Example 1: Imperotoninnano-formulations: They were made by two methods and evaluated
for formulation characteristics using the data that for Imperatonin the mass to Mol rati5 o
is70g/mol and dose is 100μmol= 27mg
[0069] The first formulation was prepared by a modified solvent evaporation technique.
Different drug to polymer ratios were used in this method (1:2, 1:3, 1:5). Briefly, 30 mg of PVA
was dissolved in 10 mL water. Drug was dispersed in 10 mL acetone. The dispersed extract in
10 acetone was then added dropwise to PVA solution on a magnetic stirrer (5MLH-DX, Remi
Equipments Pvt. Ltd., Bangalore). The resulting mixture was kept on the magnetic stirrer to
evaporate the organic solvent by air drying. This suspension was homogenized using high speed
homogenizer at 8496 g for 15 min, then sonicated using probe sonicator at 60 watts and 5s
interval for 10 min. It was then centrifuged at 531 g for half an h at room temperature. The
15 supernatant was separated by centrifugation at 41000 g for 30 min in a cooling ultracentrifuge at
4 °C (Sigma 3K30). Imperatoninnano form appeared as sediment which was separated and resuspended
in water. It was freeze dried by adding 2% mannitol. Nanoparticles thus formed were
evaluated for practical yield, particle size and zeta potential. Different batches of formulations
were prepared with different variables and optimized.
20 Zeta Potential:The freshly prepared NPs were diluted in the distilled water and the mixture was
sonicated during 2 min to separate the bigger aggregates. The analysis was carried out at a
scattering angle of 90° and at a temperature of T = 298.15 K. The Z-average diameter and
polydispersity index were calculated.
Particle Size Determination: The nanoparticles sizes were 198nm, 340nm, 191nm, and 282nm.
25 TEM: The nanoparticle size observed by TEM, correlated well with the particle size distribution
measured by Mastersizer.
Drug Efficiency and Drug Loading: Freshly prepared NPs solution was centrifuged at 6000
rpm/min for 20 min to sediments of solid NPs. Next, the solution of drug in the supernatant was
analyzed, assuming that drug not present in the supernatant was capsulated into PLGA NP by
30 HPLC.
- 15 -
Example- 2: Preparation of PLGA Nanoparticles containing Costunolide:
[0070] Costunolide is a naturally occurring sesquiterpene lactone, first isolated in
Saussureacostus roots, is a potent inducer of apoptosis, a potent anti-cancer agent, has antiinflammatory,
anti-viral, anti-fungal, antimycobacterial activity.
[0071] The costunolide loaded nanoparticles were fabricated by modified oil in water sing5 le
emulsion solvent evaporation technique. Typically, a solution of 21mg of PLGA in 3.5ml of
ethyl acetate containing costunolide( 16.6% w/w of polymer ), which was suitably stirred to
ensure that all material was dissolved. An aqueous phase containing TWEEN80 as stabilizer
(86mg of TWEEN80 in 10ml of Milli-Q-water; 0.86% w/v) which was homogenized for 1min
10 by vortex. The organic phase 2ml was then added to 10ml of an aqueous phase. Then sonicated
using a microtip probe sonicator set at 50W of energy output (XL 2002 Sonicator ultrasonic
liquid processor) during 4min to produce the oil-in-water emulsion. The formed o/w emulsion
was stirred at room temperature (22oC) by a magnetic stirrer at 800rpm for two-hours to
evaporate organic solvent. The nanoparticles were recovered by ultracentrifugation (13,500rpm,
15 15min, Hitachi). The amount of non-entrapped costunolide in the supernatant was determined by
HPLC. The nanoparticles were washed twice with water in order to remove the adsorbed
costunolide. The washing solutions were eliminated by a further centrifugation as described
above. The purified nanoparticles were freeze-dried. Costunolide loaded (15-25%, w/w of
polymer) nanoparticles were prepared by dissolving both; costunolide and polymer, in different
20 organic solvents ethyl acetate (EA), acetone (ACE), and dichloromethane (DCM). The organic
phase was then added to an aqueous phase containing PVA as stabilizer, keeping rest of the
method same except probe sonication time. Sonication time was varied between 1 and 4min,
increase in the sonication time leads to a reduction in the nanoparticle diameter.
[0072] The foregoing examples are merely illustrative and are not to be taken as limitations
25 upon the scope of the invention. Various changes and modifications to the disclosed
embodiments will be apparent to those skilled in the art. Such changes and modifications may be
made without departing from the scope of the invention.
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ADVANTAGES OF THE PRESENT INVENTION
[0073] The present invention provides anticancer formulations for safe herbal materials with
enhanced absorption.
[0074] The present invention provides PLGA linked nanoparticles of Imperatorin, exhibiting
enhanced anti-cancer activity5 .
[0075] The present invention provides formulation of nanoparticles of Imperatorin using
PLGA, with increased target specification

We Claim:
1. A method of preparation of PLGA based nanoparticles of Imperatorin, using dispersion
of pre-formed polymers by solvent evaporation method.
2. The nanoparticles of Imperatorin, as claimed in claim 1, prepared by the 5 method
comprising the following steps:
a) Dissolving Imperatorin in organic solvent and adding to a solution of chosen
surfactant in water;
b) Stirring the resulting mixture to evaporate the organic solvent by air drying;
10 c) Homogenising the suspension;
d) Sonicating the suspension;
e) Centrifugation followed by separation of supernatant;
f) Separating the nanoparticles as sediments and resuspending them in water; and
g) Freeze drying the suspension to obtain nanoparticles of Imperatorin.
15 3. The PLGA based nanoparticles of Imperatorin, as claimed in claim 1, having anti-cancer
activity with enhanced absorption.
4. The PLGA based nanoparticles of Imperatorin, as claimed in claim 1,having increased
target specification.