Description:FIELD OF INVENTION:
The present invention relates to the field of hydrogels. Particularly, the present invention relates to a hydrogel composition and a process for the preparation thereof.

BACKGROUND OF THE INVENTION:
Hyaluronic acid (HA) is a linear mucopolysaccharide composed of repeating disaccharide units of ß(1,4)-glucoronicacid(GlcUA) and ß(1,3)-N-acetylglucosamine (GlcNAC) present in all the vertebrates and also in small number of microbial pathogens. Due toits bio-compatible nature and high moisture retention capacity, it is widely used in various medical, cosmetic, and food applications. Hyaluronic acid is widely used in the design of engineered hydrogel, due to its bio-functionality, as well as numerous sites for modification with reactive groups. The present study focused on proving its anti-aging property through various experimental in-vitro and ex vivo studies. The cell line uptake study shows the distribution of HA-Cht hydrogel in HaCaT cell line. Cell cytotoxicity and antioxidant study performed to show the toxicity levels as well as its antioxidant properties respectively. The Ex-vivo studies by skin permeation and steady state flux analysis revealed the capability of HA-CHT hydrogel to get into the skin. Drug concentration depth profile uncover the fact that the prepared hydrogel can reach the different layers of skin and helps in forming collagen. The histopathology outcomes expressively validated the enhanced penetration, diffusion, and retention of HA-CHT hydrogel for the free uptake and distribution at targeted site with negligeable local cellular toxicity offering the novel transdermal therapy of hyaluronic acid for the enhanced anti-aging effect. The ant-aging assay in-vitro exhibited that a dose dependant inhibition decreased in the order of collagenase>elastase>tyrosinase at IC50 values of 58.7, 82.5, 158.5 and µg ml-1, respectively as compared to EGCG standard. Overall results revealed that HA-CHT hydrogel exhibited higher catalytic activity against all prospective enzymes than HA.
Hyaluronic acid is a linear polysaccharide composed of repeating disaccharide units of ß(1,4)-glucoronic acid(GlcUA) and ß(1,3)-N-acetylglucosamine (GlcNAC)(Widner et al., 2005). It is a member of the glycosaminoglycan family, which includes chondritin sulphate, dermatin sulphate and heparin sulphate. Hyaluronic acid differs from the other major groups of glycosaminoglycans i.e, it does not have sulfate group. It exists in a random coil configuration, which is polyanionic at physiological pH. At high molecular weights, these random coils become entangled to form a viscoelastic gel. Hyaluronic acid possesses a unique set of characteristics: its solution manifests very unusual rheological properties and is exceedingly lubricious and hydrophilic (Saranraj et al., 2013).Hyaluronic acid (HA) is widely used in the design of engineered hydrogels, due to its biofunctionality, as well as numerous sites for modification with reactive groups.HA hydrogels are now evolving in their design to be responsive to a range of cues, to present dynamic environments, and to possess multiple functionalities such as sophisticated structures and biochemical signals. Our goal with this report is to highlight recent advances in the development of HA hydrogels and their continued application to numerous biomedical conditions (Christopher et al., 2016)
Hyaluronic acid plays an important role in biomedical applications due to its high biocompatibility nature. It is used for various joint disorders including osteoarthritis.It has been used in the synthesis of scaffolds in wound healing applications, and as anti-inflammatory agent.In tissue engineering the hyaluronic acid plays a prominent role.Atopic dermatitis can be treated with hyaluronic acid. The FDA has approved the use of hyaluronic acid during eye surgeries including cataract removal, corneal transplantaion and repair of a detached retina and other eye injuries.Hyaluronic acid is the most common ingredient used in almost all skin care products.Due to its moisture retention property it is effectively used in treating effects of skin aging.Hyaluronic acid also used to prevent the effects of aging. Infact, hyaluronic acid has been promoted as a “fountain of Youth”.
A hydrogel is a crosslinked hydrophilic polymer that does not dissolve in water. They are highly absorbent yet maintain well defined structures.These properties underpin several applications, especially in the biomedical area. Many hydrogels are synthetic, but some are derived from nature.Hydrogels are used for producing contact lenses, hygiene products and wound dressings an other commercial uses of hydrogels are in drug delivery and tissue engineering more advanced studies required in these streams as well. High production costs of hydrogels are limiting their further commercialization. These natural macromolecules are typically polysaccharides and proteins comprised of glycosidic and amino acid repeating units, respectively.
Hyaluronic acid hydrogels range from relatively static matrices to those that exhibit spatiotemporally dynamic properties through external triggers like light. Such hydrogels are being explored for the culture of cells in vitro, as carriers for cells in-vivo, or to deliver therapeutics, including in an environmentally responsive manner. The future will bring new examples of HA hydrogels due to the synthetic diversity of HA.
Hyaluronan (HA) represents one biopolymer that can be modified and processed to form hydrogels for biomedical applications . Owing to their biocompatibility, tunable properties, and native biofunctionality, hydrogels built from HA are increasingly versatile for a myriad of applications. There are now widespread examples of modified HA macromers that form either covalent or physical hydrogels through crosslinking reactions such as with click chemistry or supramolecular assemblies of guest-host pairs. HA hydrogels range from relatively static matrices to those that exhibit spatiotemporally dynamic properties through external triggers like light. Such hydrogels are being explored for the culture of cells in vitro, as carriers for cells in vivo, or to deliver therapeutics, including in an environmentally responsive manner. The future will bring new examples of HA hydrogels due to the synthetic diversity of HA.
One such hydrogel out of chitosan and hyaluronic acid combination have been prepared and employed for proving its antiaging properties. An attempt made by us to study its physiochemical properties through several techniques.

Therefore, there is a need to develop a composition for antiaging properties. The present invention provides a better solution for the same.

OBJECTIVE OF THE INVENTION:
An objective of the present invention is to provide a composition for anti-aging properties.

An objective of the present invention is to provide a process for a composition for anti-aging properties.

SUMMARY OF THE INVENTION:
Accordingly, the present invention provides a hydrogel composition comprising hyaluronic acid and chitosan gel.
In an embodiment, the present invention provides that the ratio of hyaluronic acid with chitosan is 1:1 to 1:5 w/w.
In an embodiment, the present invention provides a process for preparation of hydrogel comprising dissolving HA (1% w/v) and Chitosan (4% w/v) in 1% Aqueous acetic acid solution; stirring the solution at 1000 rpm for 8 hours at room temperature to obtain homogenous HA-CHT viscous solution.
In another embodiment, the present invention provides that the HA-CHT solution is stored at 1-8oC for 20-30h for the stabilization and for complete polymer hydration of chitosan.
In another embodiment, the present invention provides that further preparing albumin solution separately in distilled water and mixing with glycerol and Vitamin E at 400-1000 rpm for 5 h in room temperature to obtain the homogenous solution.
In another embodiment, the present invention provides that the albumin solution is then added gradually into 40 ml HA-CHT solution to obtain the hydrogel.
In another embodiment, the present invention provides that the resulting hydrogel is stirred for another 1-2 hour at room temperature for the final homogenous viscous hydrogel loaded with HA.
In another embodiment, the present invention provides that the obtained HA-CHT hydrogel was sterilized at 121oC for 25 min.

BRIEF DESCRIPTION OF DRAWINGS:
These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read concerning the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:

Fig. 1: Illustration of Cell uptake studies of Control, Free HA and HA-CHT-HGL using Hoechst33342/Mitotracker double staining method, incubating for 4 h on Normal Human keratinocyte cell line (HaCaT cell) at 1 µg/ml of concentration. (Mean ± SD, n=3), *p<0.05 and *p<0.01compared to the control group cell;

Fig. 2: Demonstration of skin localization pictures of various epidermal layer (Upper layer, Middle Layer & Lower layer), Images (Control I-VI) showing Normal saline solution treated control group. Images (HA I-VI) showing plain HA solution treatment group whereas images (HA-CHT-NGL I-VI) showing Hyaluronic acid Chitosan nanogel treatment group of porcine skin evaluation by Phase Contrast Microscopy and CLSM on 24h of exposure visualized at 100X lens magnification using light microscope (Olympus, Japan) (n=3, data expressed as average ± SD,*denotes p<0.01); and

Fig. 3: Images (HA, I-III), (HA-CHT-NGL I-III) and (Control I-III) showing histopathology and permeating potential of plain HA, Hyaluronic acid chitosan nanogel and normal saline treated porcine skin at different epidermal layer visualized at 100X lens magnification using light microscope (Olympus, Japan), p<0.01, n=3.
Further, skilled artisans will appreciate that elements in the drawings are illustrated for simplicity and may not have been necessarily been drawn to scale. Furthermore, the drawings may show only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the drawings with details that will be readily apparent to those of ordinary skill in the art having the benefit of the description herein.

DETAILED DESCRIPTION:
To promote an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.

Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase “in an embodiment”, “in another embodiment”, and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or sub-systems or elements or structures or components proceeded by "comprises...a" does not, without more constraints, preclude the existence of other devices or other sub-systems or other elements or other structures or other components or additional devices or additional sub-systems or additional elements or additional structures or additional components.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.
Embodiments of the present invention will be described below in detail concerning the accompanying drawings.

The present invention discloses a composition comprising chitosan and hydrogel for anti-aging formulation.

The present invention discloses a method of isolation of HA and cold method of chitosan hydrogel formulation loaded with organic isolated HA was successfully synthesized for topical delivery to skin care platform. This formulation technique revealed that the naturally isolated HA when loaded in biodegradable chitosan hydrogel can be used as an effective topical treatment against skin ageing at very low dose and dosing frequency. The novel HA-CHT-HGL was biologically assayed for in-vitro skin uptake and cell toxicity for evaluation of biological safe margin delivery to skin. Further the HA-CHT-HGL was analysed for ex-vivo porcine skin Franz diffusion model for penetration and permeation assay by steady state drug release, concentration depth assay on skin epidermal layer, CLSM and histology analysis for the detailed analysis of topical delivery. This present formulation and biological evaluation of HA-CHT-HGL is very simple, easy to perform, inexpensive, eco-friendly, and superior substitute to chemical synthesis against skin ageing care in clinical platform. The obtained HA-CHT-HGL was highly stable formulations, showing strong anti-ageing and antioxidant activity may be considered as a new organic biodegradable topical drug delivery carrier against skin ageing and in the field of skincare cosmetic industry.
MATERIAL & METHODS
Hydrogel Synthesis method
HA (1% w/v) and Chitosan (4% w/v) was thoroughly dissolved in 1% Aqueous acetic acid solution. The solution was stirred at 1000 rpm for 8 hours at room temperature for the complete dissolution of chitosan and production of homogenous HA-CHT viscous solution. The HA-CHT solution was stored at 4oC for 24 h for the stabilization and for complete polymer hydration of chitosan. Albumin solution (4 % w/v) was prepared separately in distilled water and mixed with glycerol (2%) and Vitamin E (2 %) were stirred at 800 rpm for 5 h in room temperature to obtain the homogenous solution. 10 ml of Albumin solution was then added gradually into 40 ml HA-CHT solution, which was still in a state of stirring to obtain the hydrogel. The resulting hydrogel was stirred for another 2 hour at room temperature for the final homogenous viscous hydrogel loaded with HA. The obtained HA-CHT hydrogel was sterilized at 121oC for 25 min.

The following examples define the invention by way of illustration which does not limit the scope of the invention.

Example: 1
1.0 IN-VITRO BIOLOGICAL ASSAY
1.1 Cell Line uptake study and distribution analysis
The normal human keratinocyte cell line (HaCaT cell) was procured from national center for cell science (NCCS) Pune. And maintained in Dulbecco’s modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin, and 100 µg/mL streptomycin (PAA Laboratories GmbH, Austria) antibiotic solution. Cell lines were grown in tissue culture flasks (75 cm2) and maintained at 5% CO2 atmosphere at 37°C. After attaining the 90% confluency, the cells were trypsinized with 0.25% trypsin EDTA solution (Sigma, USA). The cells were incubated with hyaluronic acid (HA) and hydrogel (chitosan-hyaluronic acid)(equivalent to 1 µg/ml) for 4h. Cells were washed thrice with Hanks buffered salt (HBS) solution (PAA Laboratories GmbH, Austria) and fixed with 4% formaldehyde for 10min post incubation. The cell nucleus was dual stained with Hoechst 33342 and Mitotracker (a specific mitochondrial marker) at room temperature for 10-15 min prior to observe with confocal laser scanning microscopy (CLSM: fluoview TM FV 1000, Olympus).
1.1 Cell uptake and distribution analysis
The cell uptake and distribution studies exhibited by HA and HA-CHT hydrogel demonstrated clear fluorescence signals when incubated for 4 h when compared to control group (normal saline group). The images showed by the HA and HA-CHT hydrogel when tagged with Hoechst 33342/Mitotracker double staining method measured significant intracellular uptake towards HaCaT cells on prolong exposure of incubation time. Both HA and HA-CHT hydrogel exhibited fluorescent signals significantly elaborated faster uptake, sharp distribution and elevated retention time on CLSM.
Overall HA and HA-CHT hydrogel exhibited strong tendency towards HaCaT cell line due to cationic charge of hydrogel which initiate better ionic interaction between negatively charge skin membrane. Mild acidic pH of hydrogel which mimic the skin microenvironment and better transcellular uptake due to EPR accumulation mechanism. This evident the transdermal potential of HA-CHT hydrogel towards skin delivery against ageing process.
CLSM was employed to visualize the HA and HA-CHT hydrogel cellular internalization and distribution on HaCaT cell line. Here we compare and measure the fluorescent intensity of both HA and HA-CHT-HGL to deduce the uptake and distribution capacity.
The CLSM signal of HA-CHT-HGL is noteworthy compare to free HA on prolong incubation showing better distribution with enhanced accumulation in cell nuclei and large fluorescent spots devoid of any morphological variation in cell lines, concluding expedient transdermal delivery of HA-CHT-HGL against ageing process.
EXAMPLE 2:
Cellular toxicity analysis - MTT Assay
The Normal Human keratinocyte cell line (HaCaT cell) were cultured in 96 well plate and incubated with media containing free HA and HA-CHT HGL (Hyaluronic acid chitosan hydrogel) (concentration of 0.1, 1, 10 and 20 µg/mL), negative control (cells treated with blank media, 0.1 µg/mL) and positive control (Triton X-100, 0.1 µg/mL) for comparative evaluation. The media containing the samples were articulated and cells were washed thrice with HBSS after 24 h of incubation. Subsequently, about 150 µL of MTT solution (500 µg/mL in PBS) was supplemented to each well and incubated for 4 h. Later, MTT solution was cautiously enunciated and the formazan crystals were then dissolved in 200 µL of DMSO. The optical density (OD) of the resultant solution was then measured at 492 nm using an ELISA plate reader (BioTek, USA).
1.2 Cellular toxicity analysis - MTT Assay
The in-vitro MTT assay was performed on normal human HaCaT cell line to investigate cell toxicity and cell viability of free HA and HA-CHT-HGL (0.1, 1, 10 and 20 µg/mL of individual concentration). The cell toxicity assay qualitatively showed negligible toxicity on HaCaT cell line in 24h. The MTT assay displayed notable cellular viability (> 90%) byHA and HA-CHT-HGL in 24h incubation time possessing enhance and significant cell proliferation efficacy. The HA possess mild toxicity due to bare exposure and direct contact to the HaCaT cell whereas the cell toxicity is negligible by HA-CHT-HGL compare to the HA on 24 h incubation time. The cell viability by HA is found between 89-99 % on different concentration whereas by HA-CHT-HGL it is 90-98 % (at varied range) due to optimum uptake and cellular internalization. The main reason for significant cell proliferation of HA-CHT-HGL is due to pH responsive nature and outer cationic charge, leading to adequate uptake and cell binding which results in onsite release of HA. Prolong incubation time from 12 to 24 h do not alters the cell biological morphology and does not initiates unwanted cell toxicity. Overall the HA-CHT-HGL possesses significant cell proliferation efficiency which is in the acceptable limit of biological safe margin of nano-formulation for transdermal delivery. On inter-comparison of HA and HA-CHT-HGL, the HA-CHT-HGL exhibited noteworthy proliferation efficiency compared to HA which is found statistically significant when analysed by student’s T test. The MTT outcomes suggested that the nano composite drug delivery is more beneficial than barred drug exposure, displaying selective targeting and onsite drug release.
S. No. Conc. (µg/ml) Viability ± S.D.
HA HA-CHT-HGL
1 0 100 ± 00 100 ± 00
2 0.1 99.38821 ± 1.64822 98.79372 ± 3.24821
3 1 95.27393 ± 2.53921 97.46382±2.53909
4 10 92.53492 ± 4.26285 94.36482 ± 3.51923
5 20 89.64821 ± 3.51913 90.00032 ± 1.11342

EXAMPLE 3:
Antioxidant activity (DPPH Radical Scavenging Assay)
The antioxidant activity was characterized using DPPH (2, 2 -diphenyl-2-picrylhydrazyl hydrate) assay. A stock solution of DPPH in methanol was prepared, from this 1mL of this stock solution was added to 3 mL of hydrogel solution (1gm of prepared hydrogel in 10 mL of distilled water). The mixture was shaken vigorously and allowed to stand at room temperature for 30 min. Then the absorbance was measured at 517 nm by using a UV-visible spectrophotometer. Antioxidant activity was estimated by calculating the percent inhibition by following formula:
Dpph scavenging effect (%) = (control absorbance – sample absorbance)/ control absorbance x 100.

EXAMPLE 4:
Antioxidant activity (DPPH Radical Scavenging Assay)
DPPH radical scavenging assay was investigated for the evaluation of antioxidant potential of the HA and prepared HA-CHT-HGL formulation. The purple solution containing DPPH turns yellow on addition of both HA and HA-CHT-HGL, which indicates the scavenging of free radicals and presence of antioxidant activity. The results are compiled in Table and showed that the HA-CHT-HGL showed maximum inhibition with a value of 75.16 ± 0.04. Whereas the free HA showed meagre activity of value of 39.87 ± 1.27 demonstrating better and noteworthy anti-oxidant activity of HA-CHT-HGL over free HA. The antioxidant activity of HA-CHT-HGL was 2 fold better than HA which is found significant (P<0.01). The enhanced antioxidant activity of HA-CHT-HGL is due to better penetration on tissue, diffusion, and intracellular transport mediated by pH responsive chitosan degradation and HA release at targeted site. Thus, it can be concluded that onsite release of HA revealed maximum percent inhibition.
S. No. Formulation Percent DPPH scavenging effect (%)
1 HA 77.16 ± 0.04
2 HA-CHT-HGL 39.87 ± 1.27

EXAPLE 5:
Skin permeation studies
The porcine tissue was cut into 50 µm size by using a surgical scalpel (DISPO VAN, Hindustan syringes & medical devices, Faridabad, India) and deep cleaning was done to remove the hair. The skin permeation assay of hyaluronic acid (HA) and hydrogel (HA-CHT) was evaluated by observing the samples under a UV lamp prior tagging with Rhodamine 123 dye. Further the porcine tissue was placed between the donor and acceptor compartment of the Franz Diffusion cell. Then, 1 mL of hyaluronic acid tagged with Rhodamine 123 and hydrogel(chitosan-hyaluronic acid) tagged with Rhodamine 123 was added in the donor compartment. The PBS (Phosphate buffer saline) solution (pH 5) at temperature 37 ºC was stirred as the acceptor compartment dissolution media. The samples were withdrawn after 24 h and equivalent amount of the PBS solution was replaced to maintain the optimum sink condition. The collected test samples were then analyzed for drug release by UV spectroscopy method.
2.1 Skin permeation studies (Steady state flux Analysis)
For ex-vivo skin permeation evaluation, porcine skin was used for the detailed determination of the penetration capability of HA-CHT-HGL over free HA. The porcine tissue is found to be the best model to mimic the human skin in comparison to the other species, for skin penetration studies. The permeation studies were carried out by using Franz diffusion (FD) cell with diffusion area of 3.3 cm2 and volume 60 mL using porcine tissue. The ex-vivo studies laid the foundation for the in-vitro biological evaluation to determine the anti-ageing effect of HA-CHT-HGL.
The ex-vivo penetration results using the Franz diffusion cell (Fig. 3E) against porcine tissue demonstrated the potential of HA-CHT-HGL compared to the free HA under a UV lamp. The HA-CHT-HGL showed significant permeation of about 75-85% HA discharge compared to the free HA which is exhibiting 15% -25% release in the 24 h ex-vivo experiment respectively. The HA release by HA-CHT-HGL showed almost 4 fold enhanced flux rate compared to the free HA release which was found significant (P<0.01). The significant release of HA from HA-CHT-HGL was due to the surface adsorption and surface water engulfing by chitosan in the boundary of hydrogel network. Here the steady state flux was governed by a diffusion mechanism which showed a mutual connection between diffusion constant (D) and steady state flux (J). The diffusion constant phenomenon exhibited that the increase in D is directly proportional to the J due to enhanced interference of epidermal corneocytes which results in the significant steady state flux (J). On the other hand free HA showed no significant release early surface adsorption on skin leading to drug dispersion mechanism or unbound drug in the gel network exists, which results in the low intracellular and transcellular transport. The transcellular and intracellular transport of HA-CHT-HGL is occurs via hair follicle pathway and sweat glands pathway respectively, leading to the better diffusion through stratum corneum. Here, exterior chitosan covering played important roles in intracellular transportation due to surface cationic charge, enabling a better transport and ionic interaction between negative skin membrane charges. Overall, HA-CHT-HGL demonstrated a better steady state release compared to free HA via ex-vivo steady state flux experiment.
S. No. Time (h) Drug release (%)
HA HA-CHT-NGL
1 0 0 0
2 3 2.6473 ± 0.4538 3.5438 ± 2.6943
3 6 4.1193 ± 1.4294 14.8663 ± 3.5841
4 9 5.9982 ± 0.6582 27.8895 ± 4.6756
5 12 8.0354 ± 2.2290 45.9242 ± 4.2889
6 15 12.7291 ± 1.5302 60.0007 ± 3.6248
7 18 17.6583 ± 3.2313 69.8221 ± 4.8275
8 21 20.7921 ± 2.6909 78.6734 ± 3.0091
9 24 25.8935 ± 1.5494 86.8371 ± 2.1738
EXAMPLE 6: Drug concentration-depth profile
Initially, the porcine tissue incubated with hyaluronic acid and hydrogel(HA-CHT) separately for 24h. The pre treated porcine tissue was then washed using PBS solution(pH 5) and cut into 50 µm size using cryotome. The tissue was then divided into 3 layers as upper, middle and lower epidermal layers. The retained drug from the tissue was extracted by adding 5 mL ethyl alcohol, stirred for about 10 h and centrifuged for 20 minutes. The supernatant was collected and filtered via 0.2 mm syringe filter and analyzed for the drug content by UV spectroscopy.
2.2 Drug concentration-depth profile
The concentration depth results of hydrogel (HA-CHT-HGL) and HA elaborate potent and enhanced skin epidermal tissue uptake and retention of hydrogel(HA-CHT-HGL) over free HA. The ex-vivo concentration depth experiment showed the significant diffusion of hydrogel(HA-CHT-HGL) on the entire three epidermal layers (lower, middle and upper layer) as compared to free HA. The permeation and retention potential of hydrogel(HA-CHT-HGL) in all the epidermal layers was 4 fold better compared to the free HA and found significant (P<0.01). The results showed better retention potential of hydrogel(HA-CHT-HGL) due to enhance membrane disruption and cationic charge of chitosan which enables significant ionic interaction between the positively charged chitosan layers with negatively charged cell membrane initiating competent assembly of HA-CHT-HGL to the epidermal layers.
S. No. Tissue Concentration of extracted sample (mg/ml)
HA HA-CHT-HGL
1 Upper Epidermal 1.6678 ± 0.65781 4.6471 ± 0.0592
2 Middle Epidermal 0.9682 ± 0.0096 3.6558 ± 0.0012
3 Lower Epidermal 0.4989 ± 0.7849 2.7401 ± 0.0654
2.3 Fluorescent imaging & FACS (Fluorescence-activated cell sorting) analysis
The treated tissue was collected and washed with a PBS solution (pH 5). The tissue was then sectioned by a cryotme to obtain 5 µm size pieces prior to group in three. The sectioned tissues were treated with xylene to remove retained water and solvent further placed in glass slide (DISPOVAN, NEW DELHI) for sample retention measurement. The retention efficiency at different epidermal depth was analyzed by using a fluorescent microscope (Axiovert 2000, Carl Zeiss, Germany)
2.3 Fluorescent imaging & FACS analysis
CLSM (Confocal laser scanning microscopy) Tissue Penetration Imaging
The CLSM measurement of various epidermal layers showed noteworthy diffusion and distribution of hydrogel (HA-CHT-HGL) compared to free HA in 24 h of ex-vivo experiment. The fluorescence exhibited by the hydrogel (HA-CHT-HGL) displayed illuminated uptake and distribution in all the three epidermal layers confirmed by the fluorescent microscopy and phase contrast microscopy. The fluorescence intensity showed by the free HA is exceptionally low in all the three epidermal layers in which the intensity in the middle and lower epidermal layer is extremely negligible respectively, compared to hydrogel (HA-CHT-HGL) treated tissue. The upper, middle and lower epidermal hydrogel(HA-CHT-HGL) treated tissue layer showed the fluorescence intensity of ~85-90% (upper epidermal), ~70-75% (middle epidermal) and ~60-65% (lower epidermal) respectively compared to deficit fluorescence signal of free HA ~25-30% (upper epidermal),~15-20% (middle epidermal) & ~5-10% (lower epidermal) respectively, which is found significant (P<0.01). The treated porcine tissue showed the enhanced fluorescence over free HA in all three epidermal layers on ex-vivo experimental model over 24 h of incubation. On inter-comparison of the CLSM fluorescent signals upper, middle and lower epidermal layer of free HA portrayed a low and insufficient signals compared to the PCNGL treated porcine tissues which is found insignificant (P>0.01). The CLSM results demonstrated the noteworthy permeation capacity of lower epidermal) on transdermal delivery due to the efficient diffusion through SC and exterior chitosan structure leading to the better intracellular transportation and ionic binding with skin cells.
2.4 Histopathology study
The experimental tissue sample was collected, washed with a PBS solution (pH 5) and fixed by using 10% w/v formaldehyde solution. The tissues were processed to eradicate the formaldehyde and sectioned by using microtome. The sectioned tissues were preserved and stained with hematoxylin and eosin dye and visualize under the microscope (Axiovert 2000, Carl Zeiss, Germany)
2.4 Histopathology studies
The histopathological images demonstrated the efficient penetration efficiency of the synthesized hydrogel (HA-CHT-HGL) over free HA. The histology outcomes displayed the noteworthy disruption of epidermal layer with marked disturbance and lessening in the lipid bilayers. The cationic charge of hydrogel (HA-CHT-HGL) caused the optimum ionic interaction between the skin cells and hydrogel binding. This interaction results in the pH responsive degradation of chitosan network leading to the enhanced release of HA at epidermal site. The marked disturbance in the epidermal layer clearly elaborated strong permeation and diffusion of hydrogel (HA-CHT-HGL) when compared with the free HA exhibiting minimal or negligible disruption of the epidermal layer. The free HA showed negligible disruption due to an early surface adsorption phenomenon with small signs of slackening of the lipid bilayer which is found insignificant compared to the hydrogel (HA-CHT-HGL) formulation (P>0.01). This disruption of epidermal layer by hydrogel (HA-CHT-HGL) is devoid of any erythema and swelling of SC which is a sign of the absent of local tissue toxicity and enhanced intracellular transport in 24 h ex-vivo experiment. The histopathology outcomes expressively validated the enhanced penetration, diffusion, and retention of hydrogel (HA-CHT-HGL) over free HA with significant uptake and distribution of hydrogel at targeted site with negligible local cellular toxicity offering the novel transdermal therapy of hyaluronic acid hydrogel for the enhanced anti-ageing effect in clinical platform.
EXAMPLE 7:
3.0 In vitro evaluation of anti aging assay
The anti-aging potential of the tested samples were assessed by their ability to inhibit collagenase, elastase, and tyrosinase enzyme activities using epigallocatechingallate (EGCG) as reference agent. The assay in the absence of any enzyme was considered as control value for maximum inhibition. All the reactions were performed in triplicates. Inhibition of enzyme activity was expressed as the percentage by the following formula:
Enzyme inhibition (%) = [1-(enzyme activity in the presence of test extract/ activity without test extract)] x 100
3.1 Anti collagenase activity
Inhibition of collagenase activity was performed by the spectrophotometric method. The reaction mixture contained Tricine buffer (50 µl of 50 mM and pH 7.5), tested samples (25 µl) at different concentrations (0–100 µg/ml) and (Clostridium histolyticum) collagenase enzyme (25 µl, 0.8 U/ml) was incubated for 15 min. Synthetic substrate 2-furanacryloyl-l-leucylglycyl-l-prolyl-l-alanine (FALGPA, 50 µl of 2 mM) was then added. The change in absorbance was recorded immediately at 340 nm for 5 min using micro plate reader (BioTEk Instruments Inc., USA). Then ? A340/min was calculated.
3.2 Anti elastase activity
Inhibition of elastase activity was evaluated by a spectrophotometric method. Briefly, the test samples (50 µl) at different concentrations (0–100 µg/ml) and Tris-HCl bufer (150 µL of 0.1M, pH 8.0) were incubated with the porcine pancreatic elastase (25 µl of 0.03 U/ ml) at 25°C for 15 min. The reaction was started with the addition of N-Succinyl-Ala–Ala–Ala-p-nitroanilide as a substrate (25 µl of 1 mM in Tris-HCl buffer). The change in absorbance recorded directly at 410 nm for 5 min using micro plate reader (BioTEk Instruments Inc., USA). Then ? A410/min was calculated.
3.0 In vitro evaluation of anti aging assay
Excessive exposure to ultraviolet radiation triggers the photochronical generation of ROS, that stimulates over expression of matrix metalloproteinases enzymes such as collagenase and elastase resulting in degradation of the extracellular matrix (ECM) especially collagen, and elastin. In addition, the destructive damaging effects of ROS are usually associated with the enhancement the activity of tyrosinase enzymes. The enhancement of tyrosinase, a key enzyme in the melanin synthesis leading to atypical hyperpigmentation and appearance of fine wrinkles.
The results indicated that HA and HA-CHT-HGL exhibited a dose dependent inhibition decreased in the order of collagenase>elastase>tyrosinase at IC50 values of 58.7, 82.5, 158.5 and µg ml-1, respectively for HA-CHT-HGL, and 95.8, 103.7 and194.4 respectively for HA as compared to EGCG standard. HA-CHT-HGL exhibited higher catalytic activity against all prospective enzymes than HA.
S. No. Formulation Anti-ageing efficiency (IC50)
Collagenase Elastase Tyrosinase
1 HA 58.7 82.5 158.5
2 HA-CHT-HGL 95.8 103.7 194.4
3.3 Anti tyrosinase.
Inhibition of tyrosinase was established as described in Sigma protocol. Briefly, tested samples (1 ml) at different concentration and mushroom tyrosinase (100 µl of 15 U/ml) were mixed in phosphate buffer (0.9 ml of 0.1 mM, pH 6.8) and pre-incubated for 30 min at 25 °C. The enzyme substrate, l-DOPA solution (1 ml of 1.5 mM in 0.1 M phosphate buffer pH 6.8) was added to initiate the reaction. After incubation at 25 °C for 10 min, the absorbance was recorded at 475 nm with UV spectrophotometer.
EXAMPLE 8:
4.0 Statistical analysis
The values were expressed as mean + SD. The statistical analysis was performed by employing one way ANOVA followed by DUNNET’S T- test. The obtained value will considered statistically significant, if p value is less than 0.01(P value < 0.01). For all the experiments, each group had five replicate wells. Data are expressed as mean ± S.D. from three independent experiments (*p<0.05).
, C , Claims:We Claim:

1. A hydrogel composition comprising hyaluronic acid and chitosan gel.

2. The hydrogel as claimed in claim 1, wherein the ratio of hyaluronic acid with chitosan is 1:1 to 1:5 w/w.

3. A process for preparation of hydrogel comprising:
(i) dissolving HA (1% w/v) and Chitosan (4% w/v) in 1% Aqueous acetic acid solution;
(ii) stirring the solution at 1000 rpm for 8 hours at room temperature to obtain homogenous HA-CHT viscous solution.

4. The process as claimed in claim 1, wherein the HA-CHT solution is stored at 1-8oC for 20-30h for the stabilization and for complete polymer hydration of chitosan.

5. The process as claimed in claim 1, wherein further preparing albumin solution separately in distilled water and mixing with glycerol and Vitamin E at 400-1000 rpm for 5 h in room temperature to obtain the homogenous solution.

6. The process as claimed in claim 1, wherein the albumin solution is then added gradually into 40 ml HA-CHT solution to obtain the hydrogel.
7. The process as claimed in claim 1, wherein the resulting hydrogel is stirred for another 1-2 hour at room temperature for the final homogenous viscous hydrogel loaded with HA.

8. The process as claimed in claim 1, wherein the obtained HA-CHT hydrogel was sterilized at 121oC for 25 min.