COMPOSITION OF HESPERIDIN LOADED NANOSTRUCTURED LIPID CARRIER BASED HYDROGEL

Abstract

ABSTRACT The present invention discloses an optimize hesperidin-loaded nanostructured lipid carrier (HESNLC) hydrogel for enhanced wound healing. The NLC is formulated using hot-melt homogenization followed by subsequent probe sonication method. The optimized HES-NLC prepared using glyceryl mono stearate as a solid lipid, and sesame oil as the liquid lipid exhibited mean particle size less than 200 nm, high entrapment efficiency (>60 %) and drug loading (0.280 ± 0.002 %). FT-IR and XRD studies revealed that sesame oil created defects in the crystalline nature of glyceryl mono stearate and in turn is responsible for enhanced entrapment efficiency and loading for Hesperidin. The formulated NLCs were then incorporated into Carbopol 934 (1% w/v) hydrogel (HES-NLCG). The in-vitro skin irritation study revealed that, the developed HES-NLCG is safe for topical application. Furthermore, HES-NLCG contracted the wound area significantly faster (p < 0.05) compared to the control on day 14. In addition, the developed formulation is stable for a duration of 3 months.

Specification

Description:COMPOSITION OF HESPERIDIN LOADED NANOSTRUCTURED LIPID CARRIER BASED HYDROGEL

Field of Invention
The present invention relates to the composition of hesperidin loaded nanostructured lipid carrier-based hydrogel. More particularly, a composition of hesperidin loaded nanostructured lipid carrier-based hydrogel (HES-NLCG) for improved wound healing efficacy.

Background of the Invention
Hesperidin (a flavonoid) belongs to BCS class IV drugs, having poor solubility and permeability, drug loading efficacy and limited translational potential.
Gupta P. et al., 2023 formulated and evaluated dendrimer-based hydrogel bandage for wound healing activity. A hemolysis study revealed that the formulation was biocompatible and can be used for wound healing. The therapeutic efficacy of the prepared formulation was evaluated on a full-thickness wound, using an animal model. H&E staining of the control group showed degenerated neutrophils and eosinophils, while 10% of the formulation showed wound closure, formation of the epidermal layer, and remodeling. The MT staining of the 10% formulation showed better collagen synthesis compared to the control group. In vivo results showed that the preparation had better wound contraction activity compared to the control group; after 14 days, the control group had 79 ± 1.41, while the 10% of formulation had 98.9 ± 0.42. In a nutshell, Hsp-P-Hyd 10% showed the best overall performance in amelioration of full-thickness wounds.
Rehman et al., (2024) formulated and evaluated Hesperidin-loaded cubogel for fullthickness wound healing. The therapeutic efficacy of the formulation assessed in the animal model showed that the wound closure rate was 98.96 ± 1.50% after 14 days post-wounding compared to 89.12 ± 2.6% in the control group suggesting superior wound contraction activity. Hence, the Hesperidin-Cubogel formulation was found to be the most effective in enhancing full-thickness wound healing.
US7229651B2, the invention provides a dietary supplement comprising at least one flavonoid source and an enzyme, that is effective for inhibiting in vivo platelet activity and LDL cholesterol oxidation in a mammal at a dosage of about 30 mg/Kg or less. The supplement may contain flavonoid sources found in grape seed extracts, grape skin extracts, bilberry extracts, ginkgo biloba extracts or the flavonoid quercetin. The supplement may also contain fungal proteases, acid stable proteases and bromelain. The invention further provides a method for using the dietary supplement and an article of manufacture containing the supplement. The above patent is used for different functions. In the it is related to dietary supplement comprising flavonoid sources found in grape seed extracts, grape skin extracts, bilberry extracts, ginkgo biloba extracts or the flavonoid quercetin etc.
The present invention overcomes the problems of the prior arts, in the present invention, the HES-NLCG improved the stability of the Hesperidin. In addition, it provided the sustained release for Hesperidin from the HES-NLCG. Moreover, the results of wound healing studies indicated that developed HES-NLCG provided significantly higher wound healing efficacy compared to the control.
Objectives of the Invention
The prime objective of the present invention is to provide a composition of hesperidin loaded nanostructured lipid carrier-based hydrogel (HES-NLCG) for improved wound healing efficacy.
Another object of this invention is to provide the composition of HES-NLCG formulated by hot melt emulsification followed by subsequent sonication method.
Another objective of the present invention is to provide the composition of HES-NLCG where the Glyceryl monostearate and sesame oil, is selected as the lipid phase.
Another objective of the present invention is to provide the composition of HES-NLCG where the percentage of solid: liquid lipid ratio, surfactant concentration and drug concentration are optimized using the Box-Behnken Design.
Yet another object of this invention is to provide the composition of HES-NLCG that showed that HES-NLCG be used as a potential alternative to manage the topical wounds over its conventional oral therapy.
These and other objects of the present invention will be apparent from the drawings and descriptions herein. Every object of the invention is attained by at least one embodiment of the present invention.
Summary of the Invention
In one aspect of the present invention provides a composition of hesperidin loaded nanostructured lipid carrier-based hydrogel (HES-NLCG) for improved wound healing efficacy, prepared using the hot emulsification with subsequent ultra-sonication method.
In one of the aspects, the accurately measured amount of liquid lipid Sesame oil, and Kolliphor® TPGS (as solubilizer) is added.
In one of the aspects, the present invention, the developed composition is stable for a duration of 3 months.
Brief Description of Drawings
The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure. Further objectives and advantages of this invention will be more apparent from the ensuing description when read in conjunction with the accompanying drawing and wherein:
Figure 1 illustrates the block diagram according to the preferred embodiment of the present invention.
DETAIL DESCRIPTION OF INVENTION
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".
In the following description, numerous specific details are set forth in order to provide a thorough understanding of embodiments of the present invention. It will be apparent to one skilled in the art that embodiments of the present invention may be practiced without some of these specific details.
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.
As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.
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.
The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments. Reference will now be made in detail to the exemplary embodiments of the present invention.
The present invention discloses a composition of hesperidin loaded nanostructured lipid carrier-based hydrogel (HES-NLCG) for improved wound healing efficacy, by hot melt emulsification followed by subsequent sonication method.
It is to be noted that the Sesame oil used in the present invention is a commodity and hence, value added products. Any of these essential oils can be easily procured from market as a product. As the value-added products are exempted from NBA approval requirements, and therefore, NBA permission is not required for present invention.
In describing the preferred embodiment of the present invention, reference will be made herein to like numerals refer to like features of the invention.
According to preferred embodiment of the invention, referring to Figure 1, the composition of hesperidin loaded nanostructured lipid carrier-based hydrogel (HES-NLCG) comprises of solid lipid, liquid lipid and surfactant, Furthermore, the optimized composition is incorporated in a hydrogel for successful topical delivery and improved wound healing efficacy.
According to another embodiment of the invention, the solid lipid is glyceryl monostearate, liquid lipid used is sesame oil and it is stabilized by surfactant tween 80 to overcome its poor solubility and stability.
According to another embodiment of the invention, the composition of hesperidin loaded nanostructured lipid carrier-based hydrogel (HES-NLCG) is prepared in the following steps:
• Glyceryl mono stearate is heated above its melting point;
• For melting, the accurately measured amount of liquid lipid Sesame oil (75:25), and Kolliphor® TPGS (as solubilizer) is added;
• Thereafter, the hesperidin dissolved in acetone is added into this molten lipid mixture under constant stirring;
• an aqueous phase consisting of Tween 80 (0.879 % w/v concentration) is prepared and thermoregulated to the temperature of the lipid phase;
• the molten lipid phase containing Hesperidin (75mg) is gradually introduced into the aqueous phase under constant stirring (1500 rpm for 15 mins) to produce the primary emulsion;
• it is then introduced to probe sonicator (Vibra-cell; Sonics, Mumbai, India) at 40 % amplitude for 5 min (active for 5 s with a pause of 3 s) for size reduction;
• The obtained dispersion is left aside to attain room temperature to facilitate lipid solidification and NLC formation;
The results of the FT-IR studies also depicted that in the presence of Sesame oil, the crystallinity of Glyceryl monostearate is significantly reduced compared to that of Stearic acid.
According to an experimental embodiment of the invention, the preparation of the HES-NLCG is tested for skin irritation potential using Draize's primary skin irritation test. In the present invention, the exemestane loaded nanostructured lipid carriers is successfully developed and characterized for localized transdermal delivery of exemestane
• The DEE (%) of optimized HES-NLC is 68.10±0.28 %.
• The mean particle size for optimized formulation is below 200 nm.
• Further, The HES content in HES-NLCG is found to be 98.311± 1.24 %. It could be interpreted from the study that minimal HES loss is incurred during the inclusion of HES-NLC into the hydrogel.
• The developed HES-NLCG is tested for skin irritation potential using Draize's primary skin irritation test. The results validated the safety of the developed HES-NLCG and can be used for topical application.
• The wound area of the treated groups started to contract on day 3 compared to the other treated groups, the HES-NLCG group exhibited the most pronounced healing rate on day 9, with wound contraction (%) of 50.66±0.17 and HES-PG groups showed wound contraction (%) of 39.33±0.16 on day 9 that significantly increased to become 91.8±0.21% and 71.33±0.13% at the end of the experiment (14 days) respectively. It could be observed that HES-NLCG and HES-PG groups manifested a relatively analogous healing rate to the fusidic acid cream treated group, especially on day 14.
According to another embodiment of the invention, the percentage of solid: liquid lipid ratio, surfactant concentration and drug concentration were optimized using the Box-Behnken Design, the optimized NLC exhibited the particle size of 198 nm, PDI is 0.26 and DEE 68.10 %. The HR-TEM results show the spherical shape with the size range of 150-200 nm. Furthermore, the optimized batch shows no significant change during storage for 3 months. The viscosity studies showed pseudoplastic characteristics for both the HES-PG and HESNLCG.
The in-vitro release study revealed that HES-NLCG exhibited a sustained release for HES compared to the control. Moreover, skin irritation study on the New Zealand white rabbits (a breed of rabbits) showed that the prepared gel is safe for topical application and indicated no erythema and edema in the treated skin. Furthermore, the in-vivo wound healing study on white albino rats (a breed of rats) showed that the HES-NLCG could significantly contract the wound area as compared to control after 14th day of study.
According to another embodiment of the invention, from all the aforesaid studies it can be concluded that the developed HES-NLCG is safe, effective and could be used for the treatment of wounds Hence, the invention showed that HES-NLCG could be used as a potential alternative to manage the topical wounds over its conventional oral therapy.
According to another embodiment of the invention, the prepared HES-NLCG has industrial use/application in providing the sustained release of the Hesperidin and could improve the wound healing in full-thickness excised skin. It could be used as a potential alternative to improve the wound healing over its conventional oral therapy.

According to another embodiment of the invention, the present prepared HES-NLCG has following features that sets it apart from existing prior arts:
• the results of X-RD and FTIR studies confirmed that adding sesame oil into the glyceryl monostearate produced disordered lipid matrix.
• The disordered matrix is able to load higher amount of Hesperidin (0.280 %w/w).
• Further, the optimized HES-NLC is successfully loaded into a hydrogel and characterized.
• The developed HES-NLCG showed high drug content (98.141±0.658 %).
• Furthermore, the in-vivo wound healing study on white albino rats showed that the HESNLCG could significantly contract the wound area as compared to control after 14th day of study i.e. 91.8±0.21%.

Although a preferred embodiment of the invention has been illustrated and described, it will at once be apparent to those skilled in the art that the invention includes advantages and features over and beyond the specific illustrated construction. Accordingly it is intended that the scope of the invention be limited solely by the scope of the hereinafter appended claims, and not by the foregoing specification, when interpreted in light of the relevant prior art.
, Claims:We Claim;

1. A composition of hesperidin loaded nanostructured lipid carrier-based hydrogel (HES-NLCG) comprises of a solid lipid, a liquid lipid and a surfactant, wherein the hydrogel is prepared by hot melt emulsification followed by subsequent sonication method.

2. The A composition of hesperidin loaded nanostructured lipid carrier-based hydrogel (HES-NLCG) as claimed in claim 1, wherein the solid lipid is a glyceryl monostearate, liquid lipid is a sesame oil and the surfactant is tween 80.

3. The A composition of hesperidin loaded nanostructured lipid carrier-based hydrogel (HES-NLCG) as claimed in claim 1, wherein the hydrogen is prepared in the following steps:
• Heating the Glyceryl mono stearate above its melting point;
• For melting, the accurately measured amount of liquid lipid Sesame oil (75:25), and Kolliphor® TPGS (as solubilizer) is added;
• Thereafter, dissolving the hesperidin in acetone and is added into this molten lipid mixture under constant stirring;
• an aqueous phase consisting of Tween 80 (0.879 % w/v concentration) is prepared and thermoregulated to the temperature of the lipid phase;
• the molten lipid phase containing Hesperidin (75mg) is gradually introduced into the aqueous phase under constant stirring (1500 rpm for 15 mins) to produce the primary emulsion;
• it is then introduced to probe sonicator (Vibra-cell; Sonics, Mumbai, India) at 40 % amplitude for 5 min (active for 5 s with a pause of 3 s) for size reduction;
• The obtained dispersion is left aside to attain room temperature to facilitate lipid solidification and NLC formation.

4. The A composition of hesperidin loaded nanostructured lipid carrier-based hydrogel (HES-NLCG) as claimed in claim 1, wherein the
• The DEE (%) of optimized HES-NLC is 68.10±0.28 %;
• The mean particle size for optimized formulation is below 200 nm;
• Further, The HES content in HES-NLCG is found to be 98.311± 1.24 %, it could be interpreted from the study that minimal HES loss is incurred during the inclusion of HES-NLC into the hydrogel;
• The prepared HES-NLCG is tested for skin irritation potential using Draize's primary skin irritation test, the results validated the safety of the developed HES-NLCG and can be used for topical application;
• The wound area of the treated groups started to contract on day 3 compared to the other treated groups, the HES-NLCG group exhibited the most pronounced healing rate on day 9, with wound contraction (%) of 50.66±0.17 and HES-PG groups showed wound contraction (%) of 39.33±0.16 on day 9 that significantly increased to become 91.8±0.21% and 71.33±0.13% at the end of the experiment (14 days) respectively.

5. The A composition of hesperidin loaded nanostructured lipid carrier-based hydrogel (HES-NLCG) as claimed in claim 1, wherein the the HES-NLCG and HES-PG groups provide analogous healing rate to the fusidic acid cream treated group, on day 14.

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