FIELD OF THE INVENTION
[0001] The present disclosure relates generally to topical drug delivery systems for Mimosa pudica. More specifically, the disclosure is directed to a pharmaceutical gel formulation for wound healing comprising nanoliposomes of Mimosa pudica. The disclosure further provides a process of manufacturing 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] Liposomes are spherical shaped vesicles made of a lipid bilayer that can contain a hydrophobic or hydrophilic drug or active component in their cavities. Liposome has been used as a special drug delivery system for many drugs, especially cancer drugs, and is known for being biodegradable, enhancing penetration and overcoming drug-breakdown.
[0004] Mimosa pudica is an annual plant belonging to the Fabaceae family, also known as the touch-me-not plant or sensitive plant. The plant grows like a weed and has compound leaves that respond rapidly to touch or stimulation, a property to which it owes its name. The plant has antimicrobial properties, especially antibacterial effect and effectively treats piles, diarrhea and asthma. However, much like other herbal active constituents Mimosa pudica active component delivery is a problem. There is a need to improve the topical delivery of Mimosa pudica for its wound healing application.
[0005] The inventors of the present disclosure provide a pharmaceutical gel formulation comprising nanoliposomes of Mimosa pudica for improved wound regeneration.

OBJECTS OF THE INVENTION
[0006] An object of the present disclosure is to provide a pharmaceutical formulation for Mimosa pudica for increasing its permeability for improved wound healing.
[0007] An object of the present disclosure is to provide a pharmaceutical gel formulation for wound healing comprising nanoliposomes of Mimosa pudica.
[0008] Another object of the present disclosure is to provide a pharmaceutical gel formulation for wound healing that increases hydroxyproline; and decreases superoxide dismutase and thiobarbituric acid reactive substance (TBARS) in the skin.
[0009] Yet another object of the present disclosure is to provide a process of manufacturing a pharmaceutical gel formulation for wound healing comprising nanoliposomes of Mimosa pudica.

SUMMARY OF THE INVENTION
[0010] 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.
[0011] Aspects of present disclosure intend to increase the permeability of Mimosa pudica by encapsulating it in a nanoliposomal form in a gel formulation.
[0012] In an aspect, the present disclosure provides a pharmaceutical gel formulation for wound healing comprising (a) nanoliposomes of Mimosa pudica; and (b) one or more gelling agent(s).
[0013] In an embodiment, the formulation comprises (a) nanoliposomes comprising Mimosa pudica, soy lecithin, a surfactant, and one or more hydrating agent(s); and (b) one or more gelling agent(s).
[0014] In an embodiment, the formulation comprises (a) nanoliposomes comprising Mimosa pudica, soy lecithin, a sorbitan, and one or more hydrating agent(s); and (b) one or more gelling agent(s).
[0015] In an embodiment, the formulation may comprise whole of Mimosa pudica, a part of Mimosa pudica or an extract of Mimosa pudica.
[0016] In an embodiment, the Mimosa pudica may be present in a range of about 0.1%w/w to about 0.5%w/w of the nanoliposomes.
[0017] In an embodiment, the soy lecithin may be present in a range of about 0.6%w/w to about 0.8%w/w of the nanoliposomes.
[0018] In an embodiment, the surfactant may be selected from sorbitan monooleate, sorbitan monostearate, sorbitan monolaurate, or combinations thereof. In an embodiment, the surfactant may be present in a range of about 0.01%v/w to about 0.03%v/w of the nanoliposomes.
[0019] In an embodiment, the soy lecithin may be present in a ratio of about 5:1 to about 1:1 w/w with respect to the surfactant.
[0020] In an embodiment, the hydrating agent may be selected from ethanol, methanol, or their combination. In an embodiment, the hydrating agent may be present in a range of about 7%v/v to about 10%v/v of the nanoliposomes.
[0021] In an embodiment, the nanoliposomes may be present in a range of about 1%v/w to about 5%v/w of the formulation.
[0022] In an embodiment, the gelling agent may be selected from polyacrylic acids (or carbomer), hydroxypropyl methyl cellulose (HPMC) or their combination. In an embodiment, the gelling agent may be present in a range of about 1%w/v to about 2%w/v of the formulation.
[0023] In some embodiments, the formulation further comprises one or more pharmaceutically acceptable excipient(s). In an embodiment, the gel formulation may further comprise a solvent.
[0024] In a preferred embodiment, the formulation comprises (a) nanoliposomes comprising an aqueous extract of aerial parts of Mimosa pudica in a range of about 0.1% to about 0.5% w/w of the nanoliposomes, soy lecithin in a range of about 0.6% to about 0.8% w/w of the nanoliposomes, sorbitan monooleate in a range of about 0.01% to about 0.03% v/w of the nanoliposomes, and ethanol in a range of about 7% to about 10% v/v of the nanoliposomes; and (b) carbomer in a range of about 1% to about 2% w/v of the formulation.
[0025] In an aspect, the present disclosure provides a process of manufacturing of a pharmaceutical gel formulation for wound healing comprising nanoliposomes of Mimosa pudica, wherein the process comprises the steps of: (a) mixing soy lecithin, a surfactant, and an aqueous extract of aerial parts of Mimosa pudica and forming a thin film encapsulating Mimosa pudica using a rota evaporator; (b) hydrating the film with a hydrating agent which swells the film to form vesicles; (c) ultrasonicating the vesicles to give nanoliposomes; and (d) dispersing the nanoliposomes in a gelling agent to give the formulation.
[0026] 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
[0027] 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.
[0028] Figure 1 graphically provides the particle size distribution of the nanoliposomes, prepared as per an embodiment of the present disclosure.
[0029] Figure 2 provides the histopathology report of rat skin in excision model with infected wound on day 8 for: (a) Group 1- Control; (b) Group 2- Vehicle control (cream base without drug); (c) Group 3- topically administered 2% gel formulation as per an embodiment of the present disclosure; (d) Group 4- topically administered 4% gel formulation as per an embodiment of the present disclosure; and (e) Group 5- a positive control 5 % Betadine (w/w).
[0030] Figure 3 provides the histopathology report of rat skin in excision model with infected wound on day 16 for: (a) Group 1- Control; (b) Group 2- Vehicle control (cream base without drug); (c) Group 3- topically administered 2% gel formulation as per an embodiment of the present disclosure; (d) Group 4- topically administered 4% gel formulation as per an embodiment of the present disclosure; and (e) Group 5- a positive control 5 % Betadine (w/w).
[0031] Figure 4 provides the histopathology report of rat skin in excision model with infected wound on day 21 for: (a) Group 1- Control; (b) Group 2- Vehicle control (cream base without drug); (c) Group 3- topically administered 2% gel formulation as per an embodiment of the present disclosure; (d) Group 4- topically administered 4% gel formulation as per an embodiment of the present disclosure; and (e) Group 5- a positive control 5 % Betadine (w/w).

DETAILED DESCRIPTION OF THE INVENTION
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.”
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0045] 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.
[0046] As described herein, the term ‘effective amount’ refers to the amount of the formulation required to bring about a change or improvement in a subject without side effects or overdosing.
[0047] 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.
[0048] The term, ‘nanoliposome’ as used herein refers to a liposome comprising Mimosa pudica, wherein the liposome has a particle size in nanometric range, for example in a range of about 85nm to about 90nm.
[0049] Aspects of the present disclosure are directed to development of novel nano liposomal gel formulations of Mimosa pudica (Touch-me-not) for wound healing activity.
[0050] In an embodiment, the present disclosure provides a pharmaceutical gel formulation for wound healing comprising (a) nanoliposomes of Mimosa pudica; and (b) one or more gelling agent(s).
[0051] In an embodiment, the formulation comprises (a) nanoliposomes comprising Mimosa pudica, soy lecithin, a surfactant, and one or more hydrating agent(s); and (b) one or more gelling agent(s).
[0052] In an embodiment, the formulation comprises (a) nanoliposomes comprising Mimosa pudica, soy lecithin, a sorbitan, and one or more hydrating agent(s); and (b) one or more gelling agent(s).
[0053] In an embodiment, the formulation may comprise whole of Mimosa pudica, a part of Mimosa pudica or an extract of Mimosa pudica. The plant comprises several active components and is safely encapsulated inside the liposomes and delivered at the site of action.
[0054] In an embodiment, the part or extract may be selected from the group comprising of root, leaves, shoot, fruits, rhizome, seed, stem, barks, flower, sap, bud or combinations thereof of the plant Mimosa pudica. In an embodiment, Mimosa pudica may comprise aerial parts including leaves, stem, shoot, bud, sap, flower, fruits or combinations thereof of the plant.
[0055] In an embodiment, the extract of the plant may be an aqueous extract or an alcoholic extract. In an embodiment, the extract is an alcoholic extract, wherein the alcohol is ethanol.
[0056] In a preferred embodiment, an aqueous extract of the aerial parts of the plant may be employed. In an embodiment, the extract may be prepared by any method well-known in the art.
[0057] In an embodiment, the Mimosa pudica may be present in a range of about 0.1%w/w to about 0.5%w/w of the nanoliposomes.
[0058] In an embodiment, the soy lecithin may be present in a range of about 0.6%w/w to about 0.8%w/w of the nanoliposomes.
[0059] In an embodiment, the surfactant may be selected from sorbitan monooleate, sorbitan monostearate, sorbitan monolaurate, or combinations thereof. In a preferred embodiment, the surfactant may be sorbitan monooleate.
[0060] In an embodiment, the surfactant may be present in a range of about 0.01%v/w to about 0.03%v/w of the nanoliposomes.
[0061] In an embodiment, the soy lecithin may be present in a ratio of about 5:1 to about 1:1 w/w with respect to the surfactant. In an embodiment, the soy lecithin may be present in a ratio of about 5:1 w/w with respect to the surfactant.
[0062] In an embodiment, the hydrating agent may be selected from ethanol, methanol, or their combination. In a preferred embodiment, the hydrating agent may be a 7% solution of ethanol.
[0063] In an embodiment, the hydrating agent may be present in a range of about 7%v/v to about 10%v/v of the nanoliposomes.
[0064] In an embodiment, the nanoliposomes may be present in a range of about 1%v/w to about 5%v/w of the formulation.
[0065] In an embodiment, the gelling agent may be selected from polyacrylic acids (or carbomer), hydroxypropyl methyl cellulose, or their combination.
[0066] In an embodiment, the gelling agent may be present in a range of about 1%w/v to about 2%w/v of the formulation.
[0067] In some embodiments, the formulation further comprises one or more pharmaceutically acceptable excipients(s). In an embodiment, the excipient may be selected from co-surfactant, emulsifier, thickener, emollient, preservative, moisturizer, pH adjuster, stabilizing agent, coloring agent, fragrance, or combinations thereof. However, a person skilled in the art would appreciate that any other excipient(s) can be utilized to serve the intended purpose without departing from the scope and spirit of the disclosure.
[0068] In an embodiment, the co-surfactant may be selected from polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, poloxamer 118, polyoxyethylene sorbitan monostearate, or combinations thereof. However, a person skilled in the art would appreciate that any other co-surfactant(s) can be utilized to serve the intended purpose without departing from the scope and spirit of the disclosure.
[0069] In an embodiment, the emulsifier may be selected from beeswax, emulsifying wax, lanolin, cetyl alcohol, stearyl alcohol, sodium dioctyl sulfosuccinate, glycerol monostearate, glycerol distearate, sodium lauryl sulphate, sodium monopalmitate, sodium oleate, or combinations thereof. However, a person skilled in the art would appreciate that any other emulsifier(s) can be utilized to serve the intended purpose without departing from the scope and spirit of the disclosure.
[0070] In an embodiment, the thickener may be selected from hydroxyethyl cellulose, guar gum, cetyl alcohol, stearyl alcohol, acacia, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, or combinations thereof. However, a person skilled in the art would appreciate that any other thickener(s) can be utilized to serve the intended purpose without departing from the scope and spirit of the disclosure.
[0071] In an embodiment, the emollient may be selected from aloe gel, coconut oil, propyl paraben, paraffin, cetyl palmitate, isopropyl myristate, propylene glycol, tricapryl citrate, stearyl alcohol, polyethylene glycol 4000, or combinations thereof. However, a person skilled in the art would appreciate that any other emollient(s) can be utilized to serve the intended purpose without departing from the scope and spirit of the disclosure.
[0072] In an embodiment, the preservative may be selected from propyl paraben, methyl paraben, ethyl paraben, butyl paraben, sodium metabisulfite, or combinations thereof. However, a person skilled in the art would appreciate that any other preservative(s) can be utilized to serve the intended purpose without departing from the scope and spirit of the disclosure.
[0073] In an embodiment, the moisturizer may be selected from coconut oil, aloe gel, shea butter, olive oil, or combinations thereof. However, a person skilled in the art would appreciate that any other moisturizer(s) can be utilized to serve the intended purpose without departing from the scope and spirit of the disclosure.
[0074] In an embodiment, the pH adjuster may be selected from phosphate, citrate, ascorbic acid, or combinations thereof. However, a person skilled in the art would appreciate that any other pH adjuster(s) can be utilized to serve the intended purpose without departing from the scope and spirit of the disclosure.
[0075] In an embodiment, the coloring agent may be selected from erythrosine, tartrazine, amaranth carmine, saffron, sunset yellow, brilliant blue, caramel, indigotine, or combinations thereof. However, a person skilled in the art would appreciate that any other coloring agent(s) can be utilized to serve the intended purpose without departing from the scope and spirit of the disclosure.
[0076] In an embodiment, the fragrance may be selected from mangosteen, lemon oil, rose oil, lavender, peppermint oil, menthol, or combinations thereof. However, a person skilled in the art would appreciate that any other fragrance(s) can be utilized to serve the intended purpose without departing from the scope and spirit of the disclosure.
[0077] In an embodiment, the excipient(s) may be present in a range of about 2% w/v to about 3% w/v of the formulation.
[0078] In an embodiment, the gel formulation may further comprise a solvent.
[0079] In an embodiment, the solvent may be selected from an aqueous solvent or an organic solvent. In an embodiment, the aqueous solvent may be selected from water, distilled water, de-mineralized water, or combinations thereof. In an embodiment, the organic solvent may be selected from propylene glycol, methanol, acetone, dichloromethane, ethanol, 1-propanol, ethyl acetate, methyl acetate, isobutyl acetate, formic acid, or combinations thereof.
[0080] In an embodiment, the solvent may be present in a range of about 90% v/v to about 95% v/v of the formulation.
[0081] In a preferred embodiment, the formulation comprises (a) nanoliposomes comprising an aqueous extract of aerial parts of Mimosa pudica, soy lecithin, sorbitan monooleate, and ethanol; and (b) carbomer; wherein the soy lecithin may be present in a ratio of about 5:1 w/w with respect to sorbitan monooleate.
[0082] In a preferred embodiment, the formulation comprises (a) nanoliposomes comprising an aqueous extract of aerial parts of Mimosa pudica in a range of about 0.1% to about 0.5% w/w of the nanoliposomes, soy lecithin in a range of about 0.6% to about 0.8% w/w of the nanoliposomes, sorbitan monooleate in a range of about 0.01% to about 0.03% v/w of the nanoliposomes, and ethanol in a range of about 7% to about 10% v/v of the nanoliposomes; and (b) carbomer in a range of about 1% to about 2% w/v of the formulation.
[0083] The formulation is colloidally stable and has high actives permeability. The formulation synergistically increases the permeability of Mimosa pudica which pure Mimosa pudica or other known cream based formulations of Mimosa pudica do not provide. The formulation of the present disclosure may also provide higher anti-inflammatory and analgesic activity owing to the higher permeation of active component.
[0084] The formulation is administered topically on the skin of a subject making the active component available directly at the site of action. The skin may be a fresh wound or may be a partially healed wound. In some embodiments, the formulation may also be used on healed wounds or regenerated skin to improve skin hydration.
[0085] The liposomes comprising the active are present in nanometric sizes. In some embodiments, the nanoliposomes may have an entrapment efficiency of the active of about 70-80%.
[0086] The formulation increase re-epithelization, capillary proliferation, fibroblasts deposition and migration of leucocytes. The formulation decreases degree of inflammation.
[0087] In an embodiment, the formulation of the present disclosure may be administered along with one or more other active agent(s). These active agent(s) may be for wound healing and associated conditions and may be administered topically along with the present formulation.
[0088] In an embodiment, the present disclosure provides use of a pharmaceutical gel formulation for wound healing comprising nanoliposomes of Mimosa pudica.
[0089] In another embodiment, the present disclosure provides a medicament comprising the pharmaceutical gel formulation comprising nanoliposomes of Mimosa pudica as recited above.
[0090] In an embodiment, the present disclosure provides a process of manufacturing of a pharmaceutical gel formulation for wound healing comprising nanoliposomes of Mimosa pudica, wherein the process comprises the steps of: (a) mixing soy lecithin, a surfactant, and an aqueous extract of aerial parts of Mimosa pudica and forming a thin film encapsulating Mimosa pudica using a rota evaporator; (b) hydrating the film with a hydrating agent which swells the film to form vesicles; (c) ultrasonicating the vesicles to give nanoliposomes; and (d) dispersing the nanoliposomes in a gelling agent to give the formulation.
[0091] In an embodiment, the Mimosa pudica may be present in a range of about 0.1%w/w to about 0.5%w/w of the nanoliposomes. In an embodiment, the soy lecithin may be present in a range of about 0.6%w/w to about 0.8%w/w of the nanoliposomes. In an embodiment, the surfactant may be selected from sorbitan monooleate, sorbitan monostearate, sorbitan monolaurate, or combinations thereof. In an embodiment, the surfactant may be present in a range of about 0.01%v/w to about 0.03%v/w of the nanoliposomes. In an embodiment, the hydrating agent may be present in a range of about 7%v/v to about 10%v/v of the nanoliposomes. In an embodiment, the gelling agent may be present in a range of about 1%w/v to about 2%w/v of the formulation.
[0092] In an embodiment, the present disclosure provides a method of treatment, or amelioration of a wound of a subject by administering an effective amount of the formulation as recited above.
[0093] The effective amount may be determined by a physician based on the condition of the subject, weight, age, gender, severity of the condition, or previous medical history, among others.
[0094] 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.
EXAMPLES
[0095] 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.
[0096] MATERIALS: Mimosa pudica was obtained from Vasupharma G.I.D.C., Makarpura, Vadodara. Soya lecithin, Span® 80, ethanol and Carbopol® 940 were obtained from HiMedia.
[0097] Example 1: Manufacture of gel formulation comprising nanoliposomes of Mimosa pudica
[0098] Nanoliposomes were prepared by using 0.5gms of the aqueous extract of aerial parts of Mimosa pudica, 0.85 gms of soy lecithin and 17mL of Span® 80. For the nanoliposomes soy lecithin and Span®80 were taken in a ratio 5:1 respectively. Soy lecithin, surfactant and extract of Mimosa pudica were mixed to give a thin film encapsulating Mimosa pudica using a rota evaporator. The film was hydrated using 7% alcohol that swelled the film to give vesicles. The vesicles were ultrasonicated to give the nanoliposomes.
[0099] The prepared nanoliposomes were characterized by particle size analysis & zeta potential. Figure 1 provides the size distribution of the nanoliposomes with respect to intensity (percentage) for the optimized nanoliposomes produced by the process. Optimized nanoliposomes having particle size 85.94 nm & zeta potential -18.8 mV were incorporated into the gel formulation in concentrations of 1% w/v & 2% w/v. Mimosa pudica loaded nanoliposomes were centrifuged and were dispersed in carbopol® 940 gel 2% v/w prepared in water.
[00100] Example 2: Efficacy of gel formulation in wound healing
[00101] Efficacy of the gel formulations incorporating nanoliposomes of Example 1 were evaluated in infectious wound in excision model for rats. Two formulations were tested employing 2% v/w of the nanoliposomes in carbomer gel and 4% v/w of the nanoliposomes in carbomer gel. The rats were divided into five groups: Group 1- Control; Group 2- Vehicle control (cream base without drug); Group 3- topically administered a 2% gel formulation; Group 4- topically administered a 4% gel formulation; and Group 5- a positive control 5% Betadine (w/w).
[00102] The formulations were applied externally with the help of an applicator, once daily for 20 days, 48 h after wound infection. The wound area was traced at 0, 4, 8, 12, 16, and 20 days. The mean wound regenerated area (mm2) was determined for all the groups and the results have been provided in Table 1 below. Two animals on day 8 and day 16 from each group were euthanized and remaining animals on day 21 with excess dose of thiopental sodium and the regenerated skin along with wounded area was carefully excised and a piece was homogenized. The homogenate was estimated for hydroxyproline, superoxide dismutase (SOD) and thiobarbituric acid reactive substance (TBARS). The results from day 8 have been provided in Table 2 as displayed below. Another piece of skin was stored in 10% buffered formalin for histopathology studies.
Table 1: Effect of the formulation on infected wound in excision model in rats
Groups Wound area on day 0 Mean wound regenerated area (mm2)
Day 4 Day 8 Day 12 Day 16
1 152.3±5.8 28.8±5.9 57.5±2.7 82.2±4.2 141.8±3.5
2 166.5±8.4 83.5±5.2 62.7±3.7 94.0±4.3 148.7±3.5
3 160.5±15.3 47.3±9.3*** 90.7±2.6*** 143.2±3.3*** 156.8±3.8**
4 162.7±8.7 61.3±7.2*** 144.3±4.8*** 152.7±4.0*** 158.0±2.2***
5 160.5±4.4 59.7±6.4*** 143.5±3.5*** 149.3±3.2*** 157.7±1.8***
Data expressed as mean ±SEM: ** p < 0.01, *** p < 0.001 vs. Control; One way ANOVA followed by Dunnett post test. Route of administration of drug: Topical
Table 2: Effect of the formulation on biochemical parameters of infected wound in excision model in rats on day 8
Groups Hydroxyproline
µg/g tissue SOD
Units/mg protein TBARS
µ moles/mg protein
1 430±49.0 6.03±0.3 45.6±2.2
2 427.15±11.8 6.4±0.8 44.05±5.3
3 622.4±12.0*** 5.97±0.3 26.8±0.4**
4 672.35±15.5*** 6.1±0.6 20.2±2.6*
5 685.4±11.7*** 5.11±0.4 17.1±1.3**
Data expressed as mean ± SEM: * p < 0.05, ** p < 0.01 *** p< 0.001 vs. Control; Unpaired Student’s t test
[00103] The results of the histopathology of regenerated skin on day 8 are given in Table 3 and Figures 2(a-e). The data reveal a faster epithelization and capillary proliferation with maximum degree of fibroblasts deposition, besides lesser degree of inflammation and lower extent of migration of polymorphs lymphocytes and macrophages in rats treated topically with the 4% gel formulation. The 2% gel formulation treated rats reveal lesser degree of capillary proliferation and migration of polymorphs, lymphocytes, macrophages and fibroblast when compared to 4% formulation.
Table 3: Histological features of rat skin treated with the extracts in infected wound in excision model on day 8
Treatment Degree of inflammation Degree of Polymorphs Degree of lymphocytes Degree of macrophages Degree of epithelization Degree of capillary proliferation Degree of Fibroblasts
1 ++++ ++++ ++ + - + +
2 +++ ++++ + - - + -
3 ++ ++ - - - +++ ++++
4 + + - - - +++ ++++
5 Minimal inflammation ++++ +++ ++++
Minimal - +, Moderate - ++, More than +++ - maximum, ‘-’- not determinable

[00104] The observations on day 16 given in Table 4 indicate significant change in hydroxyproline content suggesting that wound is restored towards normalcy as concentration of hydroxyproline is a measure of concentration of collagen. Higher concentration of hydroxyproline indicates faster rate of wound healing. Biochemical analysis showed increased hydroxyproline content, which is a reflection of increased cellular proliferation and thereby increased collagen synthesis. However, a significant (p<0.001 and p<0.05) decrease in the levels of lipid peroxides level was observed in all drug treated groups when compared to the control. In the present disclosure, it’s evident that lipid peroxidation and SOD levels must decrease for restoration of wound healing. Lowering of SOD levels and lipid peroxidase indicates the formulation exhibits significant wound healing activity. The hydroxypronaline content was found to be increased. The histopathology report in Table 5 and Figures 3(a-e) reveal almost completely healed lesion with minimal inflammation in 4% formulation and Betadine treated rats. Re-epithelization, capillary proliferation, fibroblasts deposition and migration of leucocytes with inflammation appear in 4% and 2% gel formulation treated rats.
Table 4: Effect of the formulation on biochemical parameters of infected wound in excision model in rats on day 16
Groups Hydroxyproline
µg/g tissue SOD
Units/mg protein TBARS
µ moles/mg protein
1 429.85±31.0 6.5±0.3 44.33±3.6
2 425.48±12.1 6.3±0.3 42.07±2.5
3 518.83±12.0 5.92±0.3 28.07±0.9***
4 566.34±11.4 5.12±0.3 23.07±1.6***
5 580.1±12.6 4.23±0.05 16.45±0.4***
Data expressed as mean ± SEM: * p < 0.05, ** p < 0.01 *** p< 0.001 vs. Control; Unpaired Student’s t test
Table 5: Histological features of rat skin treated with the extracts in infected wound in excision model on day 16
Treatment Degree of inflammation Degree of Polymorphs Degree of lymphocytes Degree of macrophages Degree of epithelization Degree of capillary proliferation Degree of Fibroblasts
1 ++ +++ + + - + +
2 ++ ++ + + - ++ +++
3 + + + + + + +++
4 Very minimal inflammation, almost completely healed
5 Very minimal inflammation, almost completely healed
Minimal - +, Moderate - ++, More than +++ - maximum

[00105] Findings similar to day 16 were noted for hydroxyproline content and lipid peroxides level on day 21. Histopathology of skin revealed moderate inflammation, migration of polymorphs, lymphocytes, macrophages, fibroblasts and capillary proliferation in control rats. 2% gel formulation treated rats showed mild inflammation, migration of fibroblasts, migration of polymorphs, lymphocytes and macrophages and minimal capillary proliferation. Completely healed wound was observed in 4% gel formulation and Betadine treated rats (Tables 6 and 7 and Figures 4(a-e)). No significant change in SOD levels is observed in all drug treated rats at tested dose levels during the experimental period.
Table 6: Effect of the formulation on biochemical parameters of infected wound in excision model in rats on day 21
Groups Hydroxyproline
µg/g tissue SOD
Units/mg protein TBARS
µ moles/mg protein
1 92.53±5.9 4.80±0.5 45.1±0.0
2 82.00±3.4 3.60±0.2 33.7±5.8
3 70.05±3.0 3.2±0.2 15.8±1.6**
4 73.5±1.9 2.82±0.05 22.4±1.0**
5 74.65±2.2 2.95±0.2 12.2±0.0***
Data expressed as mean ± SEM: * p < 0.05, ** p < 0.01 *** p< 0.001 vs. Control; Unpaired Student’s t test
Table 7: Histological features of rat skin treated with the extracts in infected wound in excision model on day 21
Treatment Degree of inflammation Degree of Polymorphs Degree of lymphocytes Degree of macrophages Degree of epithelization Degree of capillary proliferation Degree of Fibroblasts
1 ++ + + + - ++ ++
2 ++ + + + - ++ +++
3 + - + - + +++ +++
4 Healed lesion, epithelization completed
5 Completed healed lesion
Minimal - +, Moderate - ++, More than +++ - maximum

[00106] The results reveal a significant (p<0.001) wound contraction on day 4, 8, 12 and 16 in rats treated topically with the 2% and 4% gel formulation when compared to the control. Significant (p<0.001and p<0.01) increase in hydroxyproline content was seen on day 8 in rats treated with the gel formulation of 2% and 4%. However, a significant (p<0.05) decrease in the level of lipid peroxides (TBARS) was recorded in 2% and 4% formulations treated rats.
[00107] 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.

We Claims:

1. A pharmaceutical gel formulation for wound healing comprising (a) nanoliposomes comprising Mimosa pudica, soy lecithin, a surfactant, and one or more hydrating agent(s); and (b) one or more gelling agent(s).
2. The formulation as claimed in claim 1, wherein the formulation comprises whole of Mimosa pudica, a part of Mimosa pudica or an extract of Mimosa pudica.
3. The formulation as claimed in claim 2, wherein the part or extract is selected from the group comprising of root, leaves, shoot, fruits, rhizome, seed, stem, barks, flower, sap, bud or combinations thereof of the plant Mimosa pudica.
4. The formulation as claimed in claim 1, wherein the formulation comprises an aqueous extract of aerial parts of Mimosa pudica.
5. The formulation as claimed in claim 1, wherein the Mimosa pudica is present in a range of 0.1%w/w to 0.5%w/w of the nanoliposomes.
6. The formulation as claimed in claim 1, wherein the soy lecithin is present in a range of 0.6%w/w to 0.8%w/w of the nanoliposomes.
7. The formulation as claimed in claim 1, wherein the surfactant is selected from sorbitan monooleate, sorbitan monostearate, sorbitan monolaurate, or combinations thereof.
8. The formulation as claimed in claim 1, wherein the surfactant is present in a range of 0.01%v/w to 0.03%v/w of the nanoliposomes.
9. The formulation as claimed in claim 1, wherein the soy lecithin is present in a ratio of 5:1 to 1:1 w/w with respect to the surfactant.
10. The formulation as claimed in claim 1, wherein the hydrating agent is selected from ethanol, methanol, or their combination.
11. The formulation as claimed in claim 1, wherein the hydrating agent is present in a range of 7%v/v to 10%v/v of the nanoliposomes.
12. The formulation as claimed in claim 1, wherein the gelling agent is selected from polyacrylic acid (or carbomer), hydroxypropyl methyl cellulose (HPMC) or their combination and is present in a range of 1%w/v to 2%w/v of the formulation.
13. The formulation as claimed in claim 1, wherein the nanoliposomes are present in a range of 1%v/w to 5%v/w of the formulation.
14. The formulation as claimed in claim 1, wherein the formulation comprises (a) nanoliposomes comprising an aqueous extract of aerial parts of Mimosa pudica, soy lecithin, sorbitan monooleate, and ethanol; and (b) carbomer; wherein the soy lecithin is present in a ratio of 5:1 w/w with respect to sorbitan monooleate.
15. The formulation as claimed in claim 1, wherein the formulation further comprises one or more pharmaceutically acceptable excipients(s).
16. The formulation as claimed in claim 1, wherein the formulation further comprises a solvent.
17. A process of manufacturing of a pharmaceutical gel formulation for wound healing comprising nanoliposomes of Mimosa pudica, wherein the process comprises the steps of: (a) mixing soy lecithin, a surfactant, and an aqueous extract of aerial parts of Mimosa pudica and forming a thin film encapsulating Mimosa pudica using a rota evaporator; (b) hydrating the film with a hydrating agent which swells the film to form vesicles; (c) ultrasonicating the vesicles to give nanoliposomes; and (d) dispersing the nanoliposomes in a gelling agent to give the formulation.