The present disclosure relates generally to pharmaceutical formulations. More specifically, the disclosure is directed to a formulation based on phytosomes for delivery of phyto-constituents in the liver cells. It further relates to the methods of preparing the formulation.
BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the 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] Liver is a vital organ as it performs various functions in the body, including making proteins, blood clotting factors, triglycerides, cholesterol, glycogen synthesis, and bile production. Various factors such as virus, bacteria, alcohol, drugs etc. cause inflammatory changes in the liver which leads to hepatitis, followed by chronic hepatitis, fibrosis, steatosis and cirrhosis which eventually leads to liver malfunction. Liver disorders may be treated by various chemical drugs and/or herbal compositions. Chemical drugs are associated with several side-effects upon long term usage. Nearly half of the agents used in treatment of liver diseases today are natural products and their derivatives. Although natural medicine is beneficial and safe, it lacks enhanced bioavailability and proper delivery system.
[0004] A phytosome is a complex drug delivery system. They are prepared by using phospholipids which can form a complex with phyto-constituents. Phytosomes may increase the absorption and bioavailability of the active ingredients in the target cells. A number of phytosomes exist for the treatment of liver disorders. However, they suffer from the limitations of lower effectiveness, bioavailability, biosorption, poor physiochemical properties like hydrophobicity, aqueous solubility, stability and target specificity. Hence there is a need to develop an effective phytosomal formulation for liver disorders which can overcome the problems of existing phytosomes.

[0005] The inventors of the present disclosure have arrived at a formulation based on phytosomes that has improved biosorption, bioavailability, stability and target specificity for liver cells.
OBJECTS OF THE INVENTION
[0006] An object of the present disclosure is to provide a formulation for targeted
delivery of phyto-constituents in liver cells.
[0007] An object of the present disclosure is to provide a formulation for targeted
delivery of phyto-constituents for treatment of liver disorders.
[0008] Another object of the present disclosure is to provide a formulation for
hepato-targeted delivery of phyto-constituents having improved stability,
biosorption and bioavailability.
[0009] Yet another object of the present disclosure is to provide a formulation for
hepato-targeted delivery of phyto-constituents having an extended release profile.
[0010] Still another object of the present disclosure is to provide a method of
preparing a formulation for targeted delivery of phyto-constituents in the liver
cells.
SUMMARY OF THE INVENTION
[0011] 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.
[0012] The inventors of the present disclosure arrived at a formulation that improves delivery of phyto-constituents in liver cells overcoming the low bioavailability of phyto-constituents in the target site. The formulation comprises a phytosome that is coupled with lactobionic acid and further coated with a polymer.
[0013] In an aspect, the present disclosure relates to a formulation for targeted delivery of phyto-constituents in liver cells comprising:

(a) a phytosome comprising a phospholipid and a phyto-constituent;
(b) lactobionic acid coupled to the phytosome to give a coupled
lactobionic acid-phytosome; and
(c) a polymer coating on the coupled lactobionic acid-phytosome.
[0014] In an embodiment, the phyto-constituent may be selected from the group
comprising of picrosides, tannins, gallic acid, andrographolide, ascorbic acid,
zingiberine, trigonelline, silymarin, pipeline and combinations thereof. The phyto-
constituent may be extracted from Picrorhiza kurroa, Terminalia chebula,
Terminalia bellerica, Nigella sativa, Andrographis paniculata, Embellica
officinale, Zingiber officinale, Piper nigrum, Piper longum, Piper retrofractum
and combinations thereof.
[0015] In an embodiment, the phospholipid may be selected from the group comprising of lecithin or Phosphatidylcholine (PC), cholesterol, Disteroylphosphatidylethanolamine (DSPE), Phosphatidyl ethanolamine (PE) (cephalin), Phosphatidyl serine (PS), Phosphatidyl inositol (PI), Phosphatidyl Glycerol (PG), Dipalmitoyl phosphatidyl choline (DPPC), Distearoyl phosphatidyl choline (DSPC), Dipalmitoyl phosphatidyl ethanolamine (DPPE), Dipalmitoyl phosphatidyl serine (DPPS), Dipalmitoyl phosphatidic acid (DPPA), Dipalmitoyl phosphatidyl glycerol (DPPG), Dioleoylphosphatidyl ethanolamine (DOPE), Dioleoyl phosphatidyl choline (DOPC), Dioleoyl phosphatidyl glycerol (DOPG) and combinations thereof.
[0016] In an embodiment, the polymer coating may comprise of polyvinyl pyrrolidone, cross-linked polyvinylpyrrolidone (crospovidone), cross-linked carboxymethyl cellulose (croscarmellose), cross-linked starch, sodium starch glycolate, sago starch, isphagula husk, calcium silicate, soy polysaccharides or combinations thereof.
[0017] In another aspect, the present disclosure relates to a method of preparing a formulation for targeted delivery of phyto-constituents in liver cells comprising the steps of:
(a) preparing a phytosome comprising a phospholipid and a phyto-constituent;

(b) coupling the phytosome with lactobionic acid to give a coupled
lactobionic acid-phytosome; and
(c) coating the coupled lactobionic acid-phytosome with a polymer coating
to give the formulation.
[0018] In yet another aspect, the present disclosure relates to a method of
prevention, treatment or amelioration of liver disorders in a subject by
administering a therapeutically effective amount of the formulation.
[0019] In still another aspect, the present disclosure relates to use of a
therapeutically effective amount of the formulation on a subject having liver
disorders.
[0020] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The following drawings form 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.
Figure 1: is a fluorescence microscopy graph of cancer cell line showing uptake
of (A) uncoupled phytosome and (B) formulation (immediate-release) as per an
embodiment of the present disclosure.
Figure 2: is a SEM photograph of the formulation (immediate-release) as
granules prepared by fluid bed method as per an embodiment of the present
disclosure.
Figure 3: depicts the effect of the formulation, as per an embodiment of the
present disclosure, on liver pathologic analysis by Masson's stain after CCU
treatment in rats. (A) Normal control group; (B) CCU-treated control group; (C)
CCU- and low dose of 300 mg/kg formulation-treated group; (D) CCU- and high
dose of 500 mg/kg formulation-treated group; and (E) CCU- and silymarin (200
mg/kg) of the formulation-treated group.

Figure 4: shows the Phase-Contrast Micrographs of Cultured HSCs. Isolated HSCs were cultured in the absence of formulation (A, D) or presence of formulation 200 mg/ml. (B, E) or formulation 300 mg/ml (C, F) as per an embodiment of the present disclosure. (Original magnification 200)
DETAILED DESCRIPTION OF THE INVENTION
[0022] 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 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.
[0023] 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 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. [0024] Reference throughout this specification to "one embodiment" or "an embodiment" 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.
[0025] In some embodiments, numbers have been used for quantifying weights, percentages, ratios, and so forth, to describe and claim certain embodiments of the invention and 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 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 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. [0026] 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.
[0027] 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. Also, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise. [0028] 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 to."
[0029] 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. [0030] All methods described herein can be performed in any suitable order 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 invention. [0031] 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 of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.
[0032] 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 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.
[0033] It should also be appreciated that the present disclosure can be implemented in 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. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention. [0034] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments. [0035] The following discussion provides many example embodiments of the inventive 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 include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0036] The term 'therapeutically effective amount' refers to the amount of the formulation which when administered to a subject leads to effective treatment, prophylaxis or amelioration of liver disorders without any side-effects. [0037] The term, "subject" as used herein refers to an animal, preferably a mammal, and most preferably a human. The term "mammal" used herein refers to warm-blooded vertebrate animals of the class 'mammalia' , including humans, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young, the term mammal includes animals such as cat, dog, rabbit, bear, fox, wolf, monkey, deer, mouse, pig and human. [0038] In an embodiment, the present disclosure relates to a formulation for targeted delivery of phyto-constituents in liver cells comprising:
(a) a phytosome comprising a phospholipid and a phyto-constituent;
(b) lactobionic acid coupled to the phytosome to give a coupled
lactobionic acid-phytosome; and
(c) a polymer coating on the coupled lactobionic acid-phytosome.
[0039] In an embodiment, the phyto-constituent may be selected from the group
comprising of picrosides, tannins, gallic acid, andrographolide, ascorbic acid,
zingiberine, trigonelline, silymarin, piperine and combinations thereof. In an
embodiment, the phyto-constituent may further include a solvent. In a preferred
embodiment, the solvent may be water.
[0040] In an embodiment, the phyto-constituent may be extracted from Picrorhiza kurroa, Terminalia chebula, Terminalia bellerica, Nigella sativa, Andrographis paniculata, Embellica officinale, Zingiber officinale, Piper nigrum, Piper longum, Piper retrofractum or combinations thereof.
[0041] In an embodiment, the phyto-constituent may be picrosides from Picrorhiza kurroa rhizomes, tannins and gallic acid from Terminalia chebula, tannins and gallic acid from Terminalia bellerica rhizomes, Nigella sativa seeds, andrographolide from whole plant of Andrographis paniculata, tannins and ascorbic acid from Embellica officinale, zingiberine from Zingiber officinale, trigonelline, silymarin, and piperine.

[0042] In an embodiment, the phyto-constituent may be a phyto-composition. In
an embodiment, the phyto-composition may comprise of picrosides, tannins,
gallic acid, andrographolide, ascorbic acid, zingiberine, trigonelline, silymarin,
and piperine. In an embodiment, the phyto-composition may comprise of
picrosides from Picrorhiza kurroa, rhizomes, tannins and gallic acid from
Terminalia chebula, tannins and gallic acid from Terminalia bellerica rhizomes,
Nigella sativa seeds, andrographolide from whole plant of Andrographis
paniculata, tannins and ascorbic acid from Embellica officinale, zingiberine from
Zingiber officinale, trigonelline, silymarin, and piperine. In an embodiment, the
phyto-composition may further comprise a solvent. In a preferred embodiment,
the solvent may be water.
[0043] In an embodiment, the picrosides from Picrorhiza kurroa rhizomes may be
present in the weight range of about 115mg to about 130mg, preferably about
120mg per gram of the phyto-constituent.
[0044] In an embodiment, the tannins and gallic acid from Terminalia chebula
may be present in the weight range of about 32mg to about 50mg, preferably
about 40mg per gram of the phyto-constituent.
[0045] In an embodiment, the tannins and gallic acid from Terminalia bellerica
rhizomes may be present in the weight range of about 32mg to about 50mg,
preferably about 40mg per gram of the phyto-constituent.
[0046] In an embodiment, the Nigella sativa seeds may be present in the weight
range of about 45mg to about 60mg, preferably about 50mg per gram of the
phyto-constituent.
[0047] In an embodiment, the andrographolides from whole plant of
Andrographis paniculata may be present in the weight range of about 36mg to
about 50mg, preferably about 40mg per gram of the phyto-constituent.
[0048] In an embodiment, the tannins and ascorbic acid from Embellica officinale
may be present in the weight range of about 35mg to about 50mg, preferably
about 40mg per gram of the phyto-constituent.

[0049] In an embodiment, the zingiberine from Zingiber officinale may be present in the weight range of about 43mg to about 60mg, preferably about 50mg per gram of the phyto-constituent.
[0050] In an embodiment, the trigonelline may be present in the weight range of about 5mg to about 20mg, preferably about lOmg per gram of the phyto-constituent.
[0051] In an embodiment, the silymarin may be present in the weight range of about 95mg to about HOmg, preferably about lOOmg per gram of the phyto-constituent.
[0052] In an embodiment, the piperine may be present in the weight range of about 7mg to about 20mg, preferably about lOmg per gram of the phyto-constituent.
[0053] The water may be present in the weight range of about 94mg to about
1 lOmg, preferably about lOOmg per gram of the phyto-constituent.
[0054] In an embodiment, the phospholipid may be selected from the group
comprising of lecithin or Phosphatidylcholine, cholesterol,
Disteroylphosphatidylethanolamine (DSPE), Phosphatidyl ethanolamine (PE) (cephalin), Phosphatidyl serine (PS), Phosphatidyl inositol (PI), Phosphatidyl Glycerol (PG), Dipalmitoyl phosphatidyl choline (DPPC), Distearoyl phosphatidyl choline (DSPC), Dipalmitoyl phosphatidyl ethanolamine (DPPE), Dipalmitoyl phosphatidyl serine (DPPS), Dipalmitoyl phosphatidic acid (DPPA), Dipalmitoyl phosphatidyl glycerol (DPPG), Dioleoylphosphatidyl ethanolamine (DOPE), Dioleoyl phosphatidyl choline (DOPC), Dioleoyl phosphatidyl glycerol (DOPG) and combinations thereof.
[0055] In an embodiment, the phospholipid may be present in the weight range of about 357mg to about 450mg, preferably about 400mg per gram of the phyto-constituent.
[0056] In an embodiment, the polymer coating may comprise of polyvinyl pyrrolidone, cross-linked polyvinylpyrrolidone (crospovidone), cross-linked carboxymethyl cellulose (croscarmellose), cross-linked starch, sodium starch

glycolate, sago starch, isphagula husk, calcium silicate, soy polysaccharides or combinations thereof.
[0057] In an embodiment, the formulation may further comprise a pharmaceutically acceptable excipient, well known in the art. Said excipient may be selected from super-disintegrating agents, coating materials, binders, compression aids, granulating agents, diluents, colorants, flavoring agents, lubricants, polymer matrix, surfactants, buffer solutions, cross-linking agents, bulking agents, disintegrants, glidants, antioxidants, pH modifiers, thickeners, stabilizers, emulsifiers, enhancers, preservatives, wetting agents and combinations thereof.
[0058] In an embodiment, the pharmaceutically acceptable excipient may be selected from the group comprising of alginates, sodium alginate, crospovidone, povidone, hypromellose phthalate, talc, isopropyl alcohol, water, phosphate buffer saline, 1- ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC), sugar beads/spheres, sucrose, cellulose diacetate, polysorbate, tween 80, magnesium aluminium silicates, microcrystalline cellulose, polyvinylpyrrolidone, polysaccharides, chitosan, heparin, xanthan gum, starch, pectin, gelatin, carrageenan, polymethacrylates, poly urethane, silicon, collagen, nylon, methyl cellulose, cellulose, polyacrylic acid, titanium dioxide, acetone, ethyl cellulose, hydroxy propyl methyl cellulose, sodium carboxymethyl cellulose, acetyl tributyl citrate, polymethyl methacrylate or combinations thereof.
[0059] In an embodiment, the formulation may be formulated as a solid, semi¬solid, liquid or aerosol. The formulation may be formulated as a tablet, capsule, granules, powder, pellet, microparticles, nanoparticles, sachet, solution, suspension, dispersion or lozenges. Preferably the formulation is in the form of granules.
[0060] In an embodiment, the formulation may be coated on a sugar pellet by fluid bed or spray coating techniques.
[0061] In an embodiment, the formulation improves the delivery of the phyto-constituents in the liver cell. Said formulation shows enhanced entrapment efficiency, bioavailability, biosorption, solubility and stability.

[0062] In an embodiment, the formulation is an immediate release formulation. The immediate release formulation shows release of up to 78.41% phytoconstituents within 2 hrs of administration.
[0063] In an embodiment, the formulation for targeted delivery of phyto-constituents in liver cells may be further coated with an enteric coating to give an enteric coated formulation. The enteric coating may comprise polymers selected from ethyl cellulose, hypromellose phthalate, cellulose diacetate or combinations thereof.
[0064] In an embodiment, the enteric coated formulation provides an extended release profile of the phyto-constituents at the site of action. In an embodiment, the formulation showed up to 93.4% release of phytoconstituents in about 18 hrs which demonstrated extended/prolonged release.
[0065] In an embodiment, the particle size of the formulation may be in the range of about 1055um to about 1070um, preferably about 1061 um. [0066] In an embodiment, the entrapment efficiency of the phyto-constituents in the formulation may be in the range of about 40% to about 60%, preferably it may be about 45±3.89%. Entrapment efficiency may be determined as follows:
Total amount of API-Total amount of un-entrapped API
% EE, % w/w = —: —— — x 100
Total amount or API
where API is active pharmaceutical ingredient.
[0067] In an embodiment, the percentage yield of the formulation may be in the
range of about 93% to about 94.2%, preferably it may be about 93.9±0.2%.
Percentage yield may be determined by the following equation:
Weight of formulation
% Yield, w/w =-r—; r^^ — x 100
Initial weight or extracts + excipients
[0068] In an embodiment, the phyto-constituent loading capacity of the
formulation may be in range of about 97.6% to about 102.1%>, preferably about
101.9±0.2%. Loading capacity of the formulation may be determined as follows:
Total amount of API-Total amount of un-entrapped API
% DL, % w/w = —- TTT^-r^ — x 100
Total amount or API+hpids
where API is active pharmaceutical ingredient.

[0069] Without being bound to theory, it is believed that bioavailability of phytosomes in the target site is increased by binding the liver cell specific lactobionic acid with the phytosomes. These phytosomes coated with a polymer coating prevent the early release of the phyto-composition and extend the release time. The present formulation is prepared in an optimized manner and all the phyto-constituents are water soluble which enhances the encapsulation efficiency and all the phyto-constituents are retained in phytosomes for longer time period. [0070] In another embodiment, the present disclosure relates to a method of preparing a formulation for targeted delivery of phyto-constituents in liver cells comprising the steps of:
(a) preparing a phytosome comprising a phospholipid and a phyto-constituent;
(b) coupling the phytosome with lactobionic acid to give a coupled lactobionic acid-phytosome; and
(c) coating the coupled lactobionic acid-phytosome with a polymer coating.
[0071] In an embodiment, the phytosome may be prepared by the steps of: (al) optionally grinding and mixing the phyto-constituent and the phospholipid to give a phyto-constituent-phospholipid mixture; (a2) evaporating the phyto-constituent-phospholipid mixture using a vacuum rotator, liquid nitrogen or combination thereof to give a dry film of the phyto-constituent-phospholipid mixture; and (a3) optionally hydrating the dry film with water to give the phytosome. [0072] In an embodiment, size of the phytosome may be reduced before coupling with lactobionic acid. Phytosome size reduction can be carried out by any appropriate method, including but not limited to, extrusion through uniform pore-size membranes, ultra-sonication, ultrasonic irradiation, freeze-thaw sonication (FTS), and pumping under high pressure through a small reaction tank. In a preferred embodiment, the size maybe reduced by bath sonication, homogenization and probe sonication.
[0073] In an embodiment, the coupling of the phytosome and the lactobionic acid may be performed by the steps of: (bl) preparing a lactobionic acid solution by

dissolving lactobionic acid in a buffer; (b2) adding the lactobionic acid solution to
the phytosome and incubating for formation of covalent linkage between the
phytosome and the lactobionic acid; and (b3) removing any uncoupled lactobionic
acid to give the coupled lactobionic acid-phytosome.
[0074] In an embodiment, the covalent linkage is formed in the presence of a
coupling agent. The coupling agent may be l-ethyl-3-(3- dimethylaminopropyl)
carbodiimide.
[0075] In an embodiment, the buffer may be selected from phosphate buffered
saline, acetate, borate, phosphate-citrate; phthalate-HCl, citrate, or glucuronate
buffers.
[0076] In an embodiment, the coupled lactobionic acid-phytosome may be coated
with a polymer coating by fluid bed coating or spray coating.
[0077] In an embodiment, the formulation obtained after coating with the polymer
coating may be dried, sifted, evaporated, or combinations thereof.
[0078] In an embodiment, the entire formulation may be coated on a sugar pellet.
[0079] In another embodiment, the formulation may be further coated with an
enteric coating to give an enteric coated formulation. The enteric coating may
comprise polymers selected from ethyl cellulose, hypromellose phthalate,
cellulose diacetate or combinations thereof.
[0080] In yet another embodiment, the present disclosure relates to a method of
prevention, treatment or amelioration of liver disorders in a subject by
administering a therapeutically effective amount of the formulation.
[0081] In an embodiment, the therapeutically effective amount of the formulation
may be decided by a medical practitioner based on the conditions of the subject.
In a preferred embodiment, the amount may be about 200 mg/kg to about 500
mg/kg per day for rats and about 30 mg/kg to about 50 mg/kg per day for humans.
[0082] In still another embodiment, the present disclosure relates to use of a
therapeutically effective amount of the formulation on a subject with liver
disorders. The liver disorders may include, but is not limited to, acute to chronic
hepatitis, fibrosis, steatosis, fatty liver disease or cirrhosis.

[0083] In an embodiment, the formulation may be used to protect the
morphology, fibrosis and necrosis of liver cells against hepato-toxicity caused by
oxidative stress.
[0084] In an embodiment, the formulation may be used to inhibit proliferation and
morphological change of Hepatic Stellate Cells.
[0085] 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 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.
[0086] 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.
[0087] All the extracts have been procured from Natural Remedies, Bangalore.
Example No. 1: Immediate-release formulation
[0088] The method of preparation of the formulation for targeted delivery of
phyto-constituents in liver cells is described below:
1.1. Preparation of phytosomes
1.2. Coupling of phytosomes with lactobionic acid
1.3. Coating of coupled lactobionic acid-phytosome with polymers.
1.1 Preparation of phytosomes- Phytosomes were made by combining phyto-constituents and phospholipids. The plant extracts/phyto-constituents/plant fractions and phospholipids used in the phyto-composition are given in Table No. 1.

Table No. 1: The plant extracts/phyto-constituents/plant fractions

s.
No. Name of herb/common name Name of the plant
extract
/fraction/phyto-
constituents Quantity of
standardized
extract
1 Picrorhiza kurroa rhizomes Picrosides (4%) 120 mg
2 Terminalia chebulal harde Tannins and gallic acid (40 %) 40 mg
3 Terminalia bellerical Baheda rhizomes Tannins and gallic acid (40%) 40 mg
4 Nigella sativa seeds - 50 mg
5 Andrographis paniculata whole plant Andrographolides (40%) 40 mg
6 Embellica officinale fruit juice Tannins 40%, ascorbic acid 3% 40 mg
7 Zingiber officinale Zingiberine 40% 50 mg
8 - Trigonelline 10 mg
9 - Silymarin 100 mg
10 - Piperine 10 mg
11 - Phospholipids 400 mg
12 Water for hydration - 100 mg
Total lg
1.1.1: Preparation of phyto-composition: All the constituents except phospholipids as given in Table 1 were taken and mixed in appropriate amounts to give the phyto-composition.
1.1.2: Mixing of herbal components with phospholipid
1.1.2(i). 0.2 g of Disteroylphosphatidylethanolamine (DSPE) and cholesterol (0.2 g) (dissolved in dichloromethane) in the ratio of 1:1 were added into the phyto-composition of 1.1.1.

1.1.2(ii). 0.6 g: 0.06 g of PC-DSPE mixture dissolved in chloroform: methanol (2:1) was added into the mixture of 1.1.2(i) to give the phyto-composition-phospholipid mixture. 1.1.3: Evaporation of solvents:
1.1.3(i). Vacuum rotator evaporation method- The phyto-composition-phospholipid mixture was collected in a round bottom flask and evaporation was performed in a vacuum rotary evaporator at 45°C. Evaporation produced a thin dry film of the phyto-composition-phospholipid mixture in the round bottom flask.
1.1.3(H). Complete evaporation by liquid nitrogen- The phyto-composition-phospholipid mixture was exposed to nitrogen gas flow overnight at room temperature for complete removal of solvents. 1.1.4: Hydration :
100 mg (For batch size 2 g) of distilled water was added for hydration to the dry film in a rotary at 45°C for 3 hour to give the phytosome. 1.1.5: Size reduction
To increase the biosorption phytosomal size reduction was performed. The size was reduced by bath sonication, homogenization and probe sonication. 1.2: Coupling the phytosome with lactobionic acid
This was done by coupling phytosomes with liver cell specific lactobionic acid. Lactobionic acid was attached on the surface of phytosomes using following method:
Binding of lactobionic acid with Disteroylphosphatidylethanolamine (DSPE) -1.2.(i). Preparation of lactobionic acid solution: lactobionic acid (5 mg) was dissolved in 10 ml phosphate buffer saline (PBS-pH 7.4).
1.2.(ii). Addition - 10 ml of above lactobionic acid solution was dissolved in 2 ml of phytosomal suspension.
1.2.(Hi). Addition of l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) -lOmg of EDC was added per ml of the above mixture. EDC acts as a coupling agent.

1.2.(iv). Incubation-The mixture was then incubated at room temperature for 3h
for the formation of covalent amide linkage between lactobionic acid and
disteroylphosphatidylethanolamine (DSPE) present on phytosomal surface.
1.2.(v). Removal of excessive unbound lactobionic acid- The unbound lactobionic
acid was removed by passing the dispersion through Sephadex G-50 column to
give the coupled lactobionic acid-phytosomes(LA-PS).
1.3. Preparation of spray-coated formulation:
1.3.1: Preparation of sugar-based placebo granules:
1.3. l(i). Preparation of binder solution- 670 g Pharmagrade (PG) sugar (#24 pass
by using microniser with 0.5 mm screen), 10 g of starch was dissolved in 1 liter of
purified water in a container. The solution was mixed for 10 minutes with a
homogenizer and filtered through #40 mesh into a container to form the binder
solution (Quantity produced 1.5 liters)
1.3.1(ii). Preparation of sugar beads- 3500 g Pharma grade sugar #40/50 pellets
were taken into coating pan and were loaded with PG Sugar binder solution by
spraying. The pan was rotated for 30 minutes after loading.
1.3.1(iii). Drying of loaded pellets- Above loaded pellets were collected into trays
uniformly and dried at specified temperature (40-50 ° C) for about 20 hours.
1.3.1(iv). Sifting of dried pellets to obtain granules- The above dried pellets were
collected and sifted.
1.3. l(v). Re-sifting of dried pellets- the sifted pellets were resifted to get good
fraction (quantity) of the required size granules (Quantity produced 5 kg approx.).
1.3.2: Preparation of spray-coated formulation granules-
Hepato-target granules were prepared by two methods: i). Fluid bed or ii). Spray
coating
1.3.2(i). Mixing coupled LA-PS and crospovidone XL-10- Equal quantities of
coupled lactobionic acid-phytosomes (LA-PS) and crospovidone XL-10 were
mixed with gloved hand in a blender bowl. To the mixture another equivalent
quantity of LA-PS was added and mixed with the help of gloved hand. Further
remaining quantity of LA-PS was loaded into the blender and mixed for 10
minutes.

1.3.2(H). Addition - 3g of isopropyl alcohol, 0.275 g of PVP K-30 and 0.024 g of
tween 80 were taken into the container and stirred for 10 minutes. The solution
was filtered through nylon cloth into another container.
1.3.2(iii). Coating of sugar-based placebo granules with LA-PS-crospovidone XL-
10- Coating was done by the following methods:
-Coating of sugar-based placebo granules with LA-PS-crospovidone XL-10 by
fluid bed method- The cylinder drum of pharmaceutical fluidized bed Wruster
coater coating device was charged with dummy sugar-based placebo granules (2
kg) to be coated. On to the sugar pellets LA-PS-crospovidone XL-10 blends
prepared earlier were loaded.
-Coating of sugar-based placebo granules with LA-PS-crospovidone XL-10 by
spray coating method- In spraying method the povidone solution pan was allowed
to rotate for about 10 minutes until uniform drug loading occurred. Sufficient
volume of air was drawn through the bottom of the cylinder to suspend the mass
of particles, air flow maintained at 2 kg, and the liquid to be applied was sprayed
onto the mass at the rate of 2 liters/hr to provide uniform coating.
The temperature of the fluidizing air was balanced against the spray rate to
maintain the mass of sugar-based placebo granules at the desired level of moisture
and stickiness while the coating was built up.
1.3.2(iv). Addition to the loaded granules- approx. 3.3 gm (For batch size 15gm)
of PVP k30, isopropyl alcohol and tween 20 solutions were mixed with the coated
granules.
1.3.2(v). Drying of granules -The drug loaded granules from the pan were spread
on to the trays uniformly and dried at 60°C temperature for about 3 hrs.
1.3.2(vi). Sifting of granules - After drying the granules were sifted by using
vibro-sifter to remove fines and the uniform sized granules were collected.
1.3.2(vii). Addition of talc to above dried and sifted granules -Drug loaded
granules were loaded into the coating pan. On to the drug-loaded granules, 0.04 g
of talc (For batch size 15g) was loaded while spraying remaining povidone
solution.

1.3.2(viii). Drying of talc loaded granules -The talc loaded granules from the pan
were spread on to the trays uniformly and dried at 60°C temperature for about
2hrs.
1.3.2(ix). Sifting of dried granules - The granules were sifted by using vibro-sifter
to remove fines and the uniform sized granules were collected.
1.3.2(x). Drying of granules- The mass was further maintained at 400° C for
approx. 30-60 minutes for ensuring drying of granules.
1.3.2(xi). Addition of colorants and flavors to the granules- Colorant-Erythrosine
and flavor- Mandarin berry were applied in order to increase patient compliance
by spraying.
Example no. 2: Study of immediate-release formulation
Characterization of uncoupled phytosomes and coupled phytosomes:
1. Percentage entrapment efficiency- Entrapment efficiency of phytosome was
determined using the Sephadex G-50 mini-column centrifugation method. The
entrapment efficiency of drug in coupled Phytosomes (45±3.89) was more than
the uncoupled phytosome (42±4.75).
2. Cell-uptake studies of uncoupled and coupled phytosomes-Cellular-uptake
studies were performed using BEL7402 HCC cell line (procured from National
Centre for Cell Science, Pune, India). The higher uptake of lactobionic acid
coupled phytosomes with higher fluorescence intensity (9856) was observed as
compared to uncoupled phytosomes with lower fluorescence intensity of 3266,
respectively (Figure 1).
3. Micrometric characterization of coupled phytosomal spray-coated granules-The micrometric properties like Bulk density (BD), Tapped density(TD), Compressibility index(CI), Hausner's ratio(HR) and bulkiness were carried out for the prepared granules. The Bulk density (BD), Tapped density (TD), Compressibility index (CI), Hausner's ratio (HR) and bulkiness of immediate release granules was noted to be 0.298 g/cc, 0.394 g/cc, 8.75%, 1.2, and 2.42, respectively.
4. SEM Analysis of the coupled phytosomal spray-coated granules- SEM analysis was performed for the granules prepared by fluid bed coating (FBC). The granules

prepared by FBC had smooth surface with minimal pores indicating the uniform coating of the granules (Figure 2). 5. Release profile of the formulation:
Table No. 2: Release profile of the immediate-release formulation

Time (minutes) Cumulative % release
0 0
15 25.79
30 37.61
45 46.11
60 49.38
90 64.72
120 89.41
Example 3: Extended-release formulation
Formation of extended drug release phytosomal granules involves following steps: Sub-coating of lactobionic acid-phytosome (LA-PS) granules: i. Coating of LA-PS-loaded granules with Cellulose diacetate polymer- The LA-PS-loaded granules of Example No. 1 were charged into fluidization basket. Cellulose diacetate polymer solution was atomized on to the materials while the air was allowed to circulate into the basket to keep the materials under fluidized state. The process of fluidization was done for 10 minutes to give LA-PS-Cellulose diacetate granules.
ii. Coating of LA-PS-Cellulose diacetate granules - Hypromellose phthalate polymer, titanium dioxide, acetone and isopropyl alcohol were taken into a container and mixed for 10 minutes at 1300 rpm and filtered through nylon cloth into another container. The cellulose diacetate coated granules were charged into fluidization basket. Hypromellose phthalate polymer solution was atomized on to the granules while the air was allowed to circulate into the basket to keep the materials under fluidized state. The process of fluidization was done for 10 minutes to give LA-PS-Cellulose diacetate-Hypromellose phthalate granules.

iii. Coating of LA-PS-Cellulose diacetate-Hypromellose phthalate granules- Ethyl cellulose, acetyl tributyl citrate, talc, IPA and acetone were taken into a container, mixed in homogenizer for 15 minutes and filtered through nylon cloth into another container. The hypromellose phthalate coated granules were charged into fluidization basket. Ethyl cellulose polymer solution was atomized on to the materials while the air was allowed to circulate into the basket to keep the materials under fluidized state. The process of fluidization was done for 10 minutes to give LA-PS-Cellulose diacetate-Hypromellose phthalate-Ethyl cellulose coated granules.
iv. Drying of LA-PS-Cellulose diacetate-Hypromellose phthalate-Ethyl cellulose coated granules- The final coated granules were dried at ambient conditions for 2 h and sifted through vibro-sifter to collect uniform sized granules. After drying the granules were sifted by using vibro-sifter to remove fines and the uniform sized granules were collected.
Table No. 3 below depicts the composition of various formulations prepared by Fluid Bed coating method.
Table No. 3: Ethyl Cellulose formulations

Ingredients for 15gms EC 1 EC 2 EC 3 EC 4 EC 5
Coupled phytosomes 6 6 6 6 6
Povidone K 30 0.275 0.275 0.275 0.275 0.275
Ethyl Cellulose 0.010 0.012 0.014 0.016 0.018
Cellulose Diacetate 0.012 0.014 0.016 0.018 0.020
Hypromellose Phthalate 0.135 0.135 0.135 0.135 0.135
Titanium Dioxide 0.015 0.015 0.015 0.015 0.015
Acetyl Tributyl Citrate 0.01 0.01 0.01 0.01 0.01
Acetone 1.1 1.308 1.308 1.308 1.308
IPA 3 3 3 3 3
Talc 0.037 0.037 0.037 0.037 0.037
Crospovidone SL 10 0.06 0.06 0.06 0.06 0.06
Sugar spheres 4.400 4.380 4.360 4.340 4.320

Purified water 1.44 1.44 1.44 1.44 1.44
Tween 80 0.024 0.024 0.024 0.024 0.024
Total Quantity 15gms
Example No. 4: Study of extended-release formulation
1. Micrometric characterization of the formulation- The micrometric properties like Bulk density (BD), Tapped density(TD), Compressibility index(CI), Hausner's ratio(HR) and bulkiness were carried out for the prepared granules, based upon these properties capsule size was estimated as shown in Table No. 4.
Table No. 4: Micrometric characterization of formulations

SNo Formulation BD (g/cc) TD (g/cc) CI (%) HR Bulkiness
1 EC-1 0.397 0.443 10.384 1.116 2.519
2 EC-2 0.386 0.431 10.441 1.117 2.591
3 EC-3 0.381 0.426 10.563 1.118 2.625
4 EC-4 0.379 0.422 10.190 1.113 2.639
5 EC-5 0.376 0.42 10.476 1.117 2.660
2. Evaluation of the formulation's activity against carbon tetrachloride (CCU)-
induced oxidative stress and hep ato-toxi city in vivo. EC-4 was studied for further
tests.
2.1 Treatment of animals- Male Wistar rats weighing 200-240 g were housed in
conventional cages with free access to water and rodent chow at 20-22°C with a
12-hour light-dark cycle. They were then divided into five groups.
All procedures involving laboratory animal use were in accordance with the
guidelines of the Instituted Animal Ethics Committee of Chitkara College of
Pharmacy, Chitkara University, Punjab, as per CPCSEA, India guidelines, for the
care and use of laboratory animals.
The experimental setting was:
1. Group A animals were treated with olive oil (1.0 mL/kg) by intraperitoneal
injection

2. Groups B-E were treated with 8% CCU/olive oil (1.0 mL/kg) by intraperitoneal injection, twice every week (Monday and Thursday) for 8 weeks.
3. Groups A and B were treated with water on Tuesday, Wednesday, Friday, and Saturday; Groups C, D and E were treated with low dose (300 mg/kg), and high dose (500 mg/kg) of the formulation, respectively, and Group F was treated with silymarin (200 mg/kg); all by oral gavage.
The mortality rate and body weight were recorded daily. At the end of the experiment, blood and organs were immediately collected after the animals were sacrificed. Liver tissue samples were taken from the left liver lobe and cut into two pieces. One piece was fixed in formalin for pathological examination. The other piece was subjected to biological analyses. The blood samples and organ tissues were all collected and stored at -80°C until use. 2.1.1 Histopathologic study:
A portion of the extracted liver was fixed in 10% formalin, processed using routine histology procedures, embedded in paraffin, cut in 5 urn sections, and mounted on a slide. The sections were then stained with hematoxylin-eosin dye and studied for histopathological changes (40 x), i.e., necrosis, fatty changes, ballooning degeneration, and lymphocyte infiltration. Masson stain was used for liver steatosis and fibrosis scoring. The formulation's exposure greatly improved liver morphological changes, fibrosis, and necrosis (refer Figure 3). 2.2 Inhibition of proliferation and morphological change of isolated Hepatic Stellate Cells (HSCs)
The in vitro study was carried out to check the direct effect of the formulation on proliferation of HSCs. It suppressed the proliferation and morphological change of HSCs (Figure 4). A, B, C, D, E and F in figure 3 are photographs numbers. Photographs A and D are with absence of formulation, Photographs B and E are with presence of formulation 200mg/ml and Photographs C and F are with formulation 300 mg/ml. HSCs were activated during monolayer culture to transform into proliferating myofibroblast like cells. The addition of the formulation at 200mg/ml clearly inhibited the activation (Figure 4). HSCs retained the spherical form that is characteristic of their quiescent state in the

presence of the formulation. In particular, it completely suppressed the
morphological change, indicating that the components of the formulation were
able to tackle the proliferation by various mechanisms.
It is clear from the above data that the present formulation was able to prevent
liver inflammation and fibrosis induced by repeated CCU administration and
oxidative stress. The effective dose of the formulation is found to be in between
200-300 mg/kg for rats and may be commensurate to approximately 50 mg/kg for
humans.
2.3 Release profile of the formulation:
Table No. 5: Extended release profile of the formulation

Time (h) Cumulative % release
0 0
1 20.44
2 33.32
4 44.76
6 51.89
8 58.23
10 68.33
12 77.23
14 80.45
16 86.55
18 93.46
[0089] The foregoing examples are merely illustrative and are not to be taken as limitations 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.

ADVANTAGES OF THE PRESENT INVENTION
[0090] The present disclosure provides a formulation that improves the targeted-
delivery of phyto-constituents in liver cells which otherwise has low
bioavailability.
[0091] The present disclosure provides a formulation having improved stability,
biosorption, and entrapment efficiency of phyto-constituents.
[0092] The present disclosure provides a formulation with an extended release
profile of phyto-constituents at the site of action.
[0093] The present disclosure provides a formulation that provides effective
treatment of liver disorders without side-effects.

We Claim:

1. A formulation for targeted delivery of phyto-constituents in liver cells
comprising:
(a) a phytosome comprising a phospholipid and a phyto-constituent;
(b) lactobionic acid coupled to the phytosome to give a coupled
lactobionic acid-phytosome; and
(c) a polymer coating on the coupled lactobionic acid-phytosome.
2. The formulation as claimed in claim 1, wherein the phyto-constituent is selected from the group comprising of picrosides, tannins, gallic acid, andrographohdes, ascorbic acid, zingiberine, trigonelline, silymarin, piperine and combinations thereof.
3. The formulation as claimed in claim 1, wherein the phyto-constituent is extracted from Picrorhiza kurroa, Terminalia chebula, Terminalia bellerica, Nigella sativa, Andrographis paniculata, Embellica officinale, Zingiber officinale, or combinations thereof.
4. The formulation as claimed in claim 1, wherein the phyto-constituent is picrosides from Picrorhiza kurroa rhizomes, tannins and gallic acid from Terminalia chebula, tannins and gallic acid from Terminalia bellerica rhizomes, Nigella sativa seeds, Andrographohdes from whole plant of Andrographis paniculata, tannins and ascorbic acid from Embellica officinale, zingiberine from Zingiber officinale, trigonelline, silymarin, and piperine.

5. The formulation as claimed in claim 4, wherein the picrosides from Picrorhiza kurroa rhizomes is present in the weight range of 115mg to 130mg per gram of the phyto-constituent.
6. The formulation as claimed in claim 4, wherein the tannins and gallic acid from Terminalia chebula are present in the weight range of 32mg to 50mg per gram of the phyto-constituent.
7. The formulation as claimed in claim 4, wherein the tannins and gallic acid from Terminalia bellerica rhizomes is present in the weight range of 32mg to 50mg per gram of the phyto-constituent.

8. The formulation as claimed in claim 4, wherein the Nigella sativa is present in the weight range of 45mg to 60mg per gram of the phyto-constituent.
9. The formulation as claimed in claim 4, wherein the Andrographolides from whole plant oi Andrographis paniculata is present in the weight range of 36mg to 50mg per gram of the phyto-constituent.

10. The formulation as claimed in claim 4, wherein the tannins and ascorbic acid from Embellica officinale are present in the weight range of 35mg to 50mg per gram of the phyto-constituent.
11. The formulation as claimed in claim 4, wherein the zingiberine from Zingiber officinale is present in the weight range of 43mg to 60mg per gram of the phyto-constituent.
12. The formulation as claimed in claim 4, wherein the trigonelline is present in the weight range of 5mg to 20mg per gram of the phyto-constituent.
13. The formulation as claimed in claim 4, wherein the silymarin is present in the weight range of 95mg to 1 lOmg per gram of the phyto-constituent.
14. The formulation as claimed in claim 4, wherein the piperine is present in the weight range of 7mg to 20mg per gram of the phyto-constituent.
15. The formulation as claimed in claim 1, wherein the phospholipid is selected from the group comprising of lecithin or Phosphatidylcholine, cholesterol, Disteroylphosphatidylethanolamine (DSPE), Disteroylphosphatidylethanolamine (DSPE), Phosphatidyl ethanolamine (PE) (cephalin), Phosphatidyl serine (PS), Phosphatidyl inositol (PI), Phosphatidyl Glycerol (PG), Dipalmitoyl phosphatidyl choline (DPPC), Distearoyl phosphatidyl choline (DSPC), Dipalmitoyl phosphatidyl ethanolamine (DPPE), Dipalmitoyl phosphatidyl serine (DPPS), Dipalmitoyl phosphatidic and combinations thereof.
16. The formulation as claimed in claim 1, wherein the polymer coating is
selected from polyvinyl pyrrolidone, cross-linked polyvinylpyrrolidone
(crospovidone), cross-linked carboxymethyl cellulose (croscarmellose), cross-
linked starch, sodium starch glycolate, sago starch, isphagula husk, calcium
silicate, soy polysaccharides or combinations thereof.

17. The formulation as claimed in claim 1, wherein particle size of the formulation
is in the range of 1055um to 1070um.
18. The formulation as claimed in claim 1, wherein the formulation further comprises a pharmaceutically acceptable excipient.
19. The formulation as claimed in claim 1, wherein the formulation is further coated with an enteric coating.
20. The formulation as claimed in claim 1, wherein the enteric coating comprises polymers selected from ethyl cellulose, hypromellose phthalate, cellulose diacetate or combinations thereof.
21. A method of preparing a formulation for targeted delivery of phyto-
constituents in liver cells comprising the steps of:
(a) preparing a phytosome comprising a phospholipid and a phyto-constituent;
(b) coupling the phytosome with lactobionic acid to give a coupled lactobionic acid-phytosome; and
(c) coating the coupled lactobionic acid-phytosome with a polymer coating.

22. The method as claimed in claim 21, wherein the phytosome is prepared by the steps of: (al) optionally grinding and mixing the phyto-constituent and the phospholipid to give a phyto-constituent-phospholipid mixture; (a2) evaporating the phyto-constituent-phospholipid mixture using a vacuum rotator, liquid nitrogen or combination thereof to give a dry film of the phyto-constituent-phospholipid mixture; and (a3) optionally hydrating the dry film with water to give the phytosome.
23. The method as claimed in claim 21, wherein size of the phytosome is reduced before coupling with lactobionic acid.
24. The method as claimed in claim 21, wherein the coupling of the phytosome and the lactobionic acid is performed by the steps of: (bl) preparing a lactobionic acid solution by dissolving the lactobionic acid in a buffer; (b2) adding the

lactobionic acid solution to the phytosome and incubating for formation of covalent linkage between the phytosome and the lactobionic acid; and (b3) removing any uncoupled lactobionic acid to give the coupled lactobionic acid-phytosome.