Claims:1. A formulation comprising nano-structured lipid carrier (NLC) particles with a core comprising of a) a solid lipid; b) a liquid lipid; c) berberine or its pharmaceutically acceptable salts; d) a cationic component and e) an emulsifying agent; wherein the core has a coating comprising zinc.
2. The formulation as claimed in claim 1, wherein the solid lipid is selected from stearic acid, glyceryl monostearate, glyceryl di-stearate or combinations thereof.
3. The formulation as claimed in claim 1, wherein the liquid lipid is selected from oleic acid, squalene, castor oil, soy lecithin, or combinations thereof.
4. The formulation as claimed in claim 1, wherein the liquid lipid is present in a weight percentage range of 1% to 5% of the formulation.
5. The formulation as claimed in claim 1, wherein the solid lipid is present in a weight percentage range of 5% to 60% of the formulation.
6. The formulation as claimed in claim 1, wherein the weight ratio of solid lipid to liquid lipid is in the range of 1:1 to 4:1.
7. The formulation as claimed in claim 1, wherein berberine is present in a weight percentage range of 1% to 10% of the formulation.
8. The formulation as claimed in claim 1, wherein the emulsifying agent is selected from polyoxyethylene ether, triblock polymers of polyoxypropylene, polysorbate, sorbitan esters, or combinations thereof.
9. The formulation as claimed in claim 1, wherein the emulsifying agent is present in a weight percentage range of 1% to 15% of the formulation.
10. The formulation as claimed in claim 1, wherein the cationic component is cholesterol or 1,2-Dioleoyloxy-3-trimethylammonium propane chloride.
11. The formulation as claimed in claim 1, wherein the cationic component is present in a weight percentage range of 1% to 5% of the formulation.
12. The formulation as claimed in claim 1, wherein the weight ratio of Berberine: solid lipid: cationic component is in the range of 1:1:1 to 4:4:4.
13. The formulation as claimed in claim 1, wherein zinc is present in a weight percentage range of 1% to 15% of the formulation.
14. A method of preparation of a formulation comprising nano-structured lipid carrier particles comprising steps of:
(a) preparing zinc oxide nanoparticles using a zinc salt and a base by precipitation method;
(b) dissolving berberine or its pharmaceutically acceptable salts, a solid lipid, a liquid lipid and a cationic component in a solvent;
(c) evaporating the solvent to give a dry lipid film;
(d) dissolving an emulsifying agent in an aqueous solvent to give an aqueous phase;
(e) melting the dry lipid film to give a lipid phase and adding the aqueous phase into the lipid phase to give a core mix;
(f) subjecting the core mix to high-speed shearing to produce a coarse dispersion; and
(g) adding the zinc oxide nanoparticles to the coarse dispersion and homogenizing to give the formulation.
15. The method as claimed in claim 14, wherein the precipitation method involves mixing of the zinc salt with the base followed by stirring and precipitation to give zinc oxide nanoparticles.
16. The method as claimed in claim 14, wherein the zinc salt is selected from zinc nitrate, zinc sulphate, zinc acetate, or combinations thereof.
17. The method as claimed in claim 14, wherein the base is selected from sodium hydroxide, potassium hydroxide, or sodium bicarbonate.

Description:FIELD OF THE INVENTION
[0001] The present disclosure relates generally to pharmaceuticals. More specifically, the disclosure is directed to a formulation comprising nano-structured lipid carrier particles comprising berberine, a solid lipid, a liquid lipid, and a surfactant, with a zinc coating. The disclosure further relates to methods of preparation of said formulations.

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] Diabetes mellitus is a metabolic disease in which the body either does not produce enough insulin or does not respond appropriately to the produced insulin. Insulin is the hormone released by pancreas responsible for absorption of sugar by the cells. In its absence, sugar remains in the blood stream and does not get absorbed into the cells resulting in high plasma concentration of sugar. Hyperglycemia is the condition wherein the sugar is present in excess in the blood stream. Diabetes mellitus is an incurable metabolic disorder that seriously threatens health. Possible treatment methods for diabetes mellitus involve available marketed formulations viz., tablets, capsules, injections etc. They however possess limitations such as, drug resistance, financial burden, toxicity, poor patient compliance and long-term medication.
[0004] Berberine is a plant alkaloid with traditional medicinal properties including weight loss, anti-microbial and anti-inflammatory properties. It is an activator of the enzyme Adenosine Monophosphate activated protein kinase (AMPK) and enhances insulin sensitivity. Berberine has been administered in oral dosage forms however they have clinical limitations like low solubility and consequent low bio-availability in the blood. There is a need to develop efficient methods of delivery of the alkaloid in the blood stream.
[0005] Nano-structured lipid carriers (NLCs) are alternate drug delivery systems to nano-particles or liposomes and were developed as a second generation lipid nano-particle to overcome the deficiencies of solid lipid nano-particles (SLNs). They comprise varying amounts of solid lipids and liquid lipids that form an amorphous matrix incorporating a lipophilic drug. However there is still need to further improve the solubility, absorption and controlled drug release by these NLCs in the body.
[0006] There is a need in the art to develop improved drug delivery systems for berberine. The inventors of the present disclosure provide a formulation incorporating berberine in zinc-coated nano-structured lipid carrier particles.

OBJECTS OF THE INVENTION
[0007] An object of the present disclosure is to provide an improved drug delivery system for berberine.
[0008] Another object of the present disclosure is to provide a formulation comprising nano-structured lipid carrier particles incorporating berberine and with a zinc-coating.
[0009] Yet another object of the present disclosure is to provide a formulation comprising nano-structured lipid carrier particles incorporating berberine with improved solubility, bioavailability and release profile.
[0010] Still another object of the present disclosure is to provide a method of preparation of a formulation comprising nano-structured lipid carrier particles incorporating berberine and with a zinc-coating.

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] NLC’s involve a core comprising solid and liquid lipids that create an asymmetric matrix for incorporation of different types and varying amounts of an otherwise insoluble, hydrophilic drug. In the present disclosure the core is further coated with a layer of zinc coating to enhance the bioavailability and release profile of the drug in the NLC.
[0013] In an aspect, the present disclosure relates to a formulation comprising nano-structured lipid carrier particles with a core comprising of a) a solid lipid; b) a liquid lipid; c) berberine or its pharmaceutically acceptable salt; d) a cationic component; and e) an emulsifying agent; wherein the core has a coating comprising zinc.
[0014] Berberine is the active ingredient that improves the insulin secretion efficiency. The solid lipid may be selected from stearic acid, glyceryl monostearate, or glyceryl di-stearate. The liquid lipid may be selected from oleic acid, squalene, castor oil, or soy lecithin. The weight ratio of solid lipid to liquid lipid may range from 1:1 to 4:1. The ingredients are chosen such that they are biocompatible and non-toxic.
[0015] The emulsifying agent may be selected from polyoxyethylene ether, triblock polymers of polyoxypropylene, or polysorbate. The cationic component may be selected from cholesterol, or 1,2-Dioleoyloxy-3-trimethylammonium propane chloride.
[0016] In another aspect, the present disclosure relates to a method of preparation of a formulation comprising nano-structured lipid carrier particles by precipitation method and hot-melt dispersion or homogenization.
[0017] In yet another aspect, the present disclosure relates to a method of preparation of a formulation comprising nano-structured lipid carrier particles comprising steps of:
(a) preparing zinc oxide nanoparticles using a zinc salt and a base by precipitation method;
(b) dissolving berberine or its pharmaceutically acceptable salt, a solid lipid, a liquid lipid and a cationic component in a solvent;
(c) evaporating the solvent to give a dry lipid film;
(d) dissolving an emulsifying agent in an aqueous solvent to give an aqueous phase;
(e) melting the dry lipid film to give a lipid phase and adding the aqueous phase into the lipid phase to give a core mix;
(f) subjecting the core mix to high-speed shearing to produce a coarse dispersion; and
(g) adding the zinc oxide nanoparticles of (a) to the coarse dispersion and homogenizing to give the formulation.
[0018] In yet another aspect, the present disclosure relates to a method of treatment, alleviation or amelioration of diabetes mellitus in a subject comprising administering a therapeutically effective amount of the formulation.
[0019] 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
[0020] 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 pictorial comparison of (A) Blank (without Berberine) Zn-coated NLC particles formulation and (B) Zn-coated Berberine NLC particles formulation as per an embodiment of the present disclosure
Figure 2: is a Transmission Electron Microscopy (TEM) showing the morphology of the NLC particles as per an embodiment of the present disclosure. 2A, 2B and 2C depict particle size distribution, shape and morphology at different magnification powers.
Figure 3: is a Scanning Electron Microscopy (SEM) showing the surface morphology of the NLC particles as per an embodiment of the present disclosure. 3A, 3B and 3C depict morphology of prepared NLCs, roughness and its dimensions.
Figure 4: is a graph showing comparative % cumulative release of berberine from control sample and the formulation as per an embodiment of the present disclosure.
Figure 5: is a comparative ex-vivo drug permeation study of control sample and the formulation as per an embodiment of the present disclosure.
Figure 6: represents a particle size distribution graph of a formulation comprising nano-structured lipid carrier particles without zinc coating.
Figure 7: is a particle size distribution graph of a formulation(F1) as per an embodiment of the present disclosure.
Figure 8: represents a zeta potential distribution of a formulation comprising nano-structured lipid carrier particles without zinc coating.
Figure 9: is a zeta potential distribution graph of a formulation(F1) as per an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION
[0021] 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.
[0022] 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.
[0023] 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.
[0024] In some embodiments, numbers have been used for quantifying amounts, 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.
[0025] 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.
[0026] 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.
[0027] 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.”
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0034] 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.
[0035] The term ‘therapeutically effective amount’ refers to an amount that is sufficient to produce the desired effects without causing side-effects.
[0036] 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.
[0037] The inventors of the present disclosure intended to develop zinc-coated nano-structured lipid carriers (Zn-NLCs) for enhancing the bioavailability and the curative effect of berberine, an anti-diabetic phyto-medicine. Zinc coating upgrades or stabilizes the structure of NLCs in the gastrointestinal tract. Apart from this, zinc is a trace element that the body has to depend on, in humans and animals. Zinc is also required for insulin synthesis and storage.
[0038] In an embodiment, the present disclosure relates to a formulation comprising nano-structured lipid carrier (NLC) particles with a core comprising of a) a solid lipid; b) a liquid lipid; c) berberine or its pharmaceutically acceptable salts; d) a cationic component and e) an emulsifying agent; wherein the core has a coating comprising zinc.
[0039] Without being bound to theory, it is believed that the formulation increases the permeation of berberine by using P-glycoprotein’s inhibitors (such as polysorbates) followed by coating of zinc onto the walls of NLCs that further provide stability to the NLCs’ structure in the acidic environment.
[0040] In an embodiment, the solid lipid may be selected from stearic acid, glyceryl monostearate, glyceryl di-stearate or combinations thereof.
[0041] In an embodiment, the solid lipid may be present in the weight percentage range of about 5% to about 60% of the formulation.
[0042] In an embodiment, the liquid lipid may be selected from oleic acid, squalene, castor oil, soy lecithin, or combinations thereof.
[0043] In an embodiment, the liquid lipid may be present in a weight percentage range of about 1% to about 5% of the formulation.
[0044] In an embodiment, the weight ratio of solid lipid to liquid lipid may be in the range from about 1:1 to about 4:1.
[0045] In an embodiment, the pharmaceutically acceptable salts include chloride, bromide, iodide, sulfate, phosphate, hydrochloride, citrate or other organic acid salts. The definition of berberine also includes it hydrates. In another embodiment, berberine may be a combination of the salts and hydrates.
[0046] In an embodiment, active ingredient berberine may be present in the weight range of about 1% to about 10% of the formulation based on solubility. Berberine being a phyto-chemical does not possess chances of resistance, toxicity or side effects unlike other pharmaceutical drugs.
[0047] In an embodiment, the emulsifying agent may be selected from polyoxyethylene ether, triblock polymers of polyoxypropylene, polysorbate, sorbitan esters, or combinations thereof. Specifically it may be tween® 80, Poloxamer 108, Poloxamer 407 or combinations thereof.
[0048] In an embodiment, the emulsifying agent may be present in a weight percentage range of about 1% to about 15% of the formulation.
[0049] In an embodiment, the cationic component may be selected from cholesterol or 1,2-Dioleoyloxy-3-trimethylammonium propane chloride. Preferably it is cholesterol.
[0050] In an embodiment, the cationic component may be present in a weight percentage range of about 1% to about 5% of the formulation.
[0051] In an embodiment, the weight ratio of berberine: solid lipid: cationic component may be in the range of about 1:1:1 to about 4:4:4.
[0052] In an embodiment, the coating may form a single layer or may be an overlap of multiple layers. The coating comprising zinc, synergistically works with the NLC particles to ensure uniform dosage and controlled release of the active ingredient for more than over about 48 hours. Zinc may be present in a weight percentage range of about 1% to about 15% of the formulation.
[0053] In an embodiment, the % entrapment efficiency (EE) of the NLCs was found in the range of about 79.8% to about 94.7%. The % EE of the formulations was found to be satisfactory; hence, the method adopted for preparation of NLC formulations was found to be suitable.
[0054] In an embodiment, the particle size distribution and zeta potential of prepared NLCs were found to be in the range of 90 nm to 100 nm and -16.9 mV to -18.4 mV, respectively. (refer Figure 7 and 9)
[0055] In an embodiment, the present disclosure provides a formulation it is a non-polluting material as it is 100% biodegradable and the Zn-BeNLCs (zinc-berberine NLCs) dispersion can be taken by patient himself/herself as per dose recommended.
[0056] In an embodiment, the formulation may be administered in a solid dosage form including powder, tablet, capsule, sachet, or granules. In an embodiment, the formulation may also be administered in a semi-solid or liquid dosage forms, including aerosols, solutions, syrups, suspensions or emulsions. In an embodiment, the formulation may preferably be administered orally, intravenously, transdermally, subcutaneously, rectally, subcutaneously, or parenterally.
[0057] In an embodiment, the formulation may further comprise any pharmaceutically acceptable excipient. Said pharmaceutically acceptable excipient includes, but is not limited to, stabilizers, preservatives, solvents, binder, buffers, flavoring agents, or coloring agents.
[0058] In an embodiment, the formulation increases the solubility and bio-availability of berberine in blood stream. It does not face the problems associated with first pass metabolism and possibility of pain.
[0059] In an embodiment, the formulation enhances the permeability of berberine across the intestinal membrane. The formulation increases the insulin secretion efficiency followed by accentuated anti-diabetic effect.
[0060] In an embodiment, the formulation is easy to apply, stable, cost-effective and eco-friendly.
[0061] In an embodiment, the lower dose of the formulation required to achieve the desired plasma concentration leads to improved patient compliance.
[0062] In an embodiment, the formulation overcomes drug expulsion, particle growth and low loading efficiency of solid lipid nano-particles.
[0063] In another embodiment, the present disclosure relates to a method of preparation of a formulation comprising nano-structured lipid carrier particles by precipitation method and hot-melt dispersion or homogenization.
[0064] In yet another embodiment, the present disclosure relates to a method of preparation of a formulation comprising nano-structured lipid carrier particles comprising steps of:
(a) preparing zinc oxide nanoparticles using a zinc salt and a base by precipitation method;
(b) dissolving berberine or its pharmaceutically acceptable salts, a solid lipid, a liquid lipid and a cationic component in a solvent;
(c) evaporating the solvent to give a dry lipid film;
(d) dissolving an emulsifying agent in an aqueous solvent to give an aqueous phase;
(e) melting the dry lipid film to give a lipid phase and adding the aqueous phase into the lipid phase to give a core mix;
(f) subjecting the core mix to high-speed shearing to produce a coarse dispersion; and
(g) adding the zinc oxide nanoparticles to the coarse dispersion and homogenizing to give the formulation.
[0065] In an embodiment of the present disclosure, the precipitation method involves mixing of zinc salt with the base followed by stirring and precipitation to give zinc oxide nanoparticles.
[0066] In an embodiment, the zinc salt may be selected from zinc nitrate, zinc sulphate, or zinc acetate. Preferably it is zinc nitrate.
[0067] In an embodiment, the base may be selected from sodium hydroxide, potassium hydroxide, or sodium bicarbonate.
[0068] In an embodiment, the solvent may be an alcohol including but not limited to methanol, ethanol, propanol, or isopropanol. In another embodiment, the aqueous solvent may be distilled water.
[0069] In an embodiment, the homogenization may be carried out by micro-fluidization, or shear emulsification.
[0070] In an embodiment, the method is easy to perform, cost-effective and industrially scalable.
[0071] In yet another embodiment, the present disclosure relates to a method of treatment, alleviation or amelioration of diabetes mellitus in a subject comprising administering a therapeutically effective amount of the formulation.
[0072] In still another embodiment, the present disclosure relates to use of the formulation for treatment, amelioration or prevention of diabetes and associated disorders in a subject.
[0073] 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.
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.
MATERIALS: Berberine Chloride was purchased from Sigma Aldrich. Cholesterol was purchased from Central drug house (P) Limited, New Delhi, India. Stearic acid was purchased from Nice chemicals Pvt. Ltd, Kerala, India. Methanol, ethanol and other solvents used in the study were purchased from SD Fine Chemicals limited, Mumbai, India. Polaxamer 188 was purchased from Sigma Aldrich. Disodium hydrogen orthophosphate, potassium dihydrogen orthophosphate, sodium hydroxide, oleic acid, zinc nitrate and potassium hydroxide were purchased from Molychem Private Limited, Mumbai, India.
Example 1: Process of preparation of the formulation
Step-I: Preparation of zinc oxide nanoparticles using precipitation method
[0074] Briefly, 1.5 M of KOH was prepared by dissolving 8.41g of KOH in 100 ml double de-ionized water in a beaker. 14.87g of ZnNO3 was dissolved in 100 ml of double distilled water (0.5 M). 0.5 M of ZnNO3 solution was transferred to the beaker containing the KOH solution, and stirred magnetically for 1 h (hour). After 1h, white suspension of ZnO nanoparticles was obtained and centrifuged at 4000 rpm. The obtained precipitates were collected and dried in hot air oven for 4 h.
Step-II; Preparation of the zinc coated nano-structured lipid carriers (Zn-NLCs) formulation by hot-melt dispersion/homogenization
[0075] Briefly, Berberine chloride, oleic acid, stearic acid and cholesterol were dissolved in an appropriate amount of methanol (90%, v/v) and then evaporated under vacuum to form a dry lipid film. The solid-lipid film was subsequently heated to melt at 60 °C. Meanwhile, two different aqueous phases containing 1% poloxamer 188 (w/v) and 1 % tweens® 80 were prepared at the same temperature and introduced into the lipid phase. After that, the mixture was subjected to high-speed shearing at 10,000 rpm for 5 minutes to produce coarse dispersions. Previously prepared zinc oxide nanoparticles (1-15% w/w) were added to the above mixture of coarse dispersion. The resulting coarse dispersions were further homogenized using a microfluidizer to obtain fine zinc coated berberine- NLCs (Zn-BBNLCs). Characterization of the obtained particles was done. Figures 2 and 3 give the TEM and SEM morphologies of the particles.
Six test formulations were prepared as per Table No. 1 below.

Table No. 1: Composition for Zn-coated berberine Nano-structured lipid carriers (Zn-BBNLCs)
Formulation code Formulation ratio (Berberine: Stearic acid: Cholesterol) % w/w/w Amount of Berberine Chloride (%w/w) Amount of stearic acid (mg) Amount of cholesterol
(mg)
F1 1:1:1 1 65 65
F2 1:1:2 2 65 130
F3 1:1:3 3 65 195
F4 1:4:1 4 260 65
F5 1:3:1 5 195 65
F6 1:2:1 10 130 65

Example 2: Studies for the formulations
2.1 The formulations F1-F6 of Example 1 were then tested for percentage cumulative drug release of berberine chloride.
In vitro release studies were performed to study the formulations. The dialysis bag diffusion technique was used to study the in vitro drug release of the formulations. Firstly, the study was carried out for the prepared formulations and then with the control. A control sample was run for comparison of drug release from the formulation and control (Control sample only consists of berberine dissolved in distilled water). Comparison was also made with a formulation comprising NLCs without any zinc coating (Test).
5ml of the formulations were placed in the dialysis bag (cellulose membrane, molecular cut off 12 – 14,000 D), hermetically sealed and immersed into 200 ml of PBS (pH 6.8) mixture. The entire system was kept at 32 ± 0.5 ºC with continuous magnetic stirring at 400 rpm/min. Samples were withdrawn at predetermined time intervals and replaced by fresh medium. The amount of drug released was determined by UV spectrophotometer at 345 nm. Control and Test were run similarly for comparison (refer Table No. 2 and Figure 4).
It is clear from the data in Table No. 2 that the cumulative release of berberine chloride from the NLCs of the formulation is more controlled than without NLCs.

Table No. 2: Percentage cumulative drug release of berberine chloride from NLCs
Sr. No Time (hr.) % cumulative release of Control (without NLCs) % cumulative release of NLCs (without Zn coating) (Test) %cumulative release of
F1 %cumulative release of
F2 % cumulative release of
F3 %cumulative release of
F4 %cumulative release of
F5 %cumulative release of
F6
1 0.25 12.04±0.002 7.65±0.0036
8.96±0.005 12.2±0.002 12.2±0.004 5.4±0.004 11.08±0.003 7.08±0.005
2 0.5 73.8±0.005
18.63±0.025 21.16±0.005 39.04±0.002 35.12±0.0011 13.88±0.001 30.64±0.003 18.27±0.001
3 1 80.28±0.002 42.56±0.056 38.28±0.002 46.52±0.003 42.4±0.003 23.28±0.001 66.76±0.001 34.88±0.003
4 2 87.5±0.004 68.92±0.036 64.44±0.001 58.64±0.004 51.36±0.001 37.52±0.002 74.28±0.002 66.28±0.002
5 4 98.12±0.005 86.58±0.098 74.62±0.003 77.64±0.002 62±0.001 66.28±0.005 83.88±0.004 72.16±.005
6 16 - - 98.36±0.001 91.07±0.004 92.36±0.004 74.2±0.002 97.64±0.001 82.28±0.001

2.2 Ex-vivo permeation studies of the drug across intestinal membrane
To check the permeability, the ileum part of the chicken intestine (collected from slaughter house) was isolated and taken for the ex-vivo permeation study. Then this tissue was thoroughly washed with physiological salt solution (PSS as permeation medium) to remove the mucous and lumen contents. The formulation (F1) approximately 1ml was injected into the ileum of the duodenum using syringe and both the sides of intestine were tightly closed. The receiver compartment filled with 200 ml of physiological salt solution with continuous aeration and a constant temperature of 37oC. Mixing was performed by means of a magnetic stirrer at 50 rpm, 5ml sample were withdrawn periodically from the receiver compartment at time intervals of 15 min, 30min, 1hr, 2hrs, 3hrs, up to 4hrs. Solution was replaced with an equal volume of fresh medium. The absorbance was measured using a UV-VIS spectrophotometer at wavelength of 345 nm, keeping the respective blanks (PSS solution). The percentage permeation for formulation was been compared with the control (refer Table No. 3 and Figure 5).
Table No. 3: ex-vivo drug Permeation study of control and NLCs formulation (F1) across intestinal membrane
Sr.
No Time
(hrs) Percentage permeation of control Percentage drug permeation of formulation (F1)
1 0.25 0.017 0.04
2 0.50 0.067 4.08
3 1 0.122 5.92
4 2 0.672 16.32
5 3 2.37 29.52
6 4 3.934 55.08

[0076] From the above tests it can be concluded that the formulation as per the present invention shows an improved drug release profile and improved permeation of Berberine across the intestinal membrane.
[0077] 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
[0078] The present disclosure provides a formulation comprising nano-structured lipid carrier particles for targeted and controlled delivery of Berberine.
[0079] The present disclosure provides a formulation comprising nano-structured lipid carrier particles that increase the bioavailability and permeability of Berberine.
[0080] The present disclosure provides a formulation that increases the insulin secretion efficiency followed by accentuated anti-diabetic effect.
[0081] The present disclosure provides a formulation that is simple, non-toxic, economic and efficient.
[0082] The present disclosure provides an economical and simple method of preparing a formulation comprising nano-structured lipid carrier particles.