DEVELOPMENT OF COSMETIC PREPARATION BEARING ESSENTIAL OILS FOR AGE DEFYING AND ANTIMICROBIAL ACTIVITY

Abstract

The present invention relates to a crosslinked chitosan/alginate dialdehyde (ADA) hydrogel incorporating nanoemulsion-based delivery systems for enhanced antimicrobial, anti-aging, and photoprotective properties, suitable for cosmeceutical and therapeutic applications. The hydrogel composition includes 20% w/w ADA as a crosslinking agent, 1% w/w calcium chloride for structural stability, 2% w/w glycerin as a humectant, 35% w/w water as the primary solvent, 4% w/w chitosan for film-forming and wound-healing effects, and 0.25% w/w andrographolide for anti-inflammatory and antimicrobial activity. The essential oil component comprises either tea tree oil (TTO) or rosemary oil (RMO) in a nanoemulsion form at concentrations of 2%, 4%, or 6% w/w to enable sustained release and enhance therapeutic efficacy. This formulation exhibits confirmed photoprotective and anti-aging benefits, providing prolonged skin protection, antimicrobial resistance, and anti-inflammatory action, ideal for applications in skincare.

Specification

Description:FIELD OF INVENTION
The present invention relates to the field of cosmetic and pharmaceutical formulations, specifically a nanoemulsion-based hydrogel designed for age-defying and antimicrobial activity using essential oils. It aims to enhance skin penetration, stability, and efficacy.
BACKGROUND OF THE INVENTION
The invention addresses the challenges in formulating an effective and stable delivery system for age-defying and antimicrobial treatments, particularly focusing on the volatile nature and solubility issues of essential oils, like Tea Tree Oil (TTO) and Rosemary Oil (RMO). Traditional delivery systems often fail to retain the stability and therapeutic efficacy of essential oils due to their rapid evaporation, poor bioavailability, and limited solubility. These issues make it difficult to deliver active compounds effectively to deeper skin layers, reducing the oils' potential benefits. To overcome this, a more advanced formulation approach is required to enhance both the stability and penetrability of essential oils, ensuring their beneficial properties are effectively harnessed for anti-aging and antimicrobial effects.
Nanoemulsions offer a promising solution to these formulation challenges. Characterized by their translucent, isotropic, and thermodynamically stable properties, nanoemulsions can disperse essential oils in a stable system, significantly enhancing their bioavailability. The nano-sized droplets, typically between 10 and 50 nm, allow for effective absorption into the skin while maintaining stability against phase separation and degradation. However, developing nanoemulsions for essential oils, especially for skin applications, presents unique challenges due to the need for non-irritating, biocompatible surfactants. Additionally, achieving a balance in the ratio of oil, water, and surfactant (Smix) is essential for creating a homogenous and transparent solution that can deliver active compounds effectively.
The invention utilizes a carefully optimized nanoemulsion-based hydrogel, employing an ideal Smix ratio to stabilize Tea Tree Oil (TTO) and Rosemary Oil (RMO). within a hydrogel matrix. This novel formulation not only addresses the volatility and instability of essential oils but also enhances their skin penetration for therapeutic benefits. Through encapsulation in a crosslinked chitosan hydrogel, the nanoemulsion ensures prolonged and controlled release of essential oils, maximizing antioxidant, antimicrobial, and anti-aging effects. By refining both the formulation and encapsulation process, this invention effectively preserves and delivers the therapeutic properties of Tea Tree Oil (TTO) and Rosemary Oil (RMO)., offering a potent solution for skin aging and microbial defense.
The following Prior art being Reported was:
IN202111054212: The present invention relates to the formulation and evaluation of a nanoemulsion-based hydrogel containing moringa seed oil for topical treatment of wounds where the M.oleifera seed oil shows a gram-positive and gram-negative bactericidal activity, indicating that it could be used to treat wound infections. In the present the ingredients used for the preparation of nanoemulsion (NEs) are oils, surfactants, co-surfactants, and additives. The present invention is differ from this as it relates to the field of cosmetic and pharmaceutical formulations, specifically a nanoemulsion-based hydrogel designed for age-defying and antimicrobial activity using essential oils
OBJECTS OF THE INVENTION
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows.
It is an object of the present disclosure to ameliorate one or more problems of the prior art or to at least provide a useful alternative
An object of the present disclosure is to provide a nanoemulsion-based hydrogel formulation that enhances skin health and therapeutic applications.Another object of the present disclosure is to.
Still another object of the present disclosure is to create a hydrogel containing nanoemulsions of essential oils, such as tea tree oil (TTO) or rosemary oil (RMO), for advanced topical treatment.
Another object of the present disclosure is to formulate the hydrogel to offer antimicrobial, anti-aging, and photoprotective effects, beneficial for both wound care and cosmeceutical applications
Still another object of the present disclosure is to use essential components including oils, surfactants, co-surfactants, and stabilizing agents to ensure stability and bioavailability.
Still another object of the present disclosure is to achieve an optimized nanoemulsion by employing a low-energy emulsification method, using a controlled ratio of oil and Smix phases with purified water for a clear, stable nanoemulsion system.
Yet another object of the present disclosure is to ensure that the nanoemulsion-based hydrogel is safe for skin application, reducing irritation compared to free oil-based hydrogels.
Yet another object of the present disclosure is to validate the hydrogel's effectiveness in deeper skin permeation and active delivery, supporting enhanced therapeutic benefits.
Yet another object of the present disclosure is to stability of essential oils within the hydrogel matrix, minimizing volatility and degradation while enabling controlled release of active compounds over extended periods.
Yet another object of the present disclosure is to exhibits desirable physical characteristics such as homogeneity, appropriate viscosity, excellent spreadability, and suitable pH, ensuring user-friendly application and consistent performance.

Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.

SUMMARY OF THE INVENTION
The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the present invention. It is not intended to identify the key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concept of the invention in a simplified form as a prelude to a more detailed description of the invention presented later.

The present invention is generally directed to the development of to nanoemulsion-based hydrogel formulations incorporating essential oils, designed for topical applications to deliver enhanced antimicrobial, anti-aging, and photoprotective effects.
An embodiment of the present inventionis Different nanoemulsion formulations encapsulating tea tree oil (TTO) and rosemary oil (RMO) were developed using the phase titration method, optimizing the oil-to-Smix ratio for stability and transparency.
Another embodiment of the invention is formulations TM6 (TTO) and RM6 (RMO) were optimized based on encapsulation efficiency, homogeneity, and particle distribution, then incorporated into a crosslinked chitosan/alginate hydrogel matrix containing acetic acid, sodium alginate, and calcium chloride to improve structural integrity and bioavailability. For comparison, free oil-based hydrogels were also prepared with TTO or RMO at 6% w/w, using glycerin as an emollient and Tween-80 as a surfactant. Characterization of the hydrogels demonstrated favorable properties, including homogeneity, pH, spreadability, and stability across various storage temperatures. Skin irritation studies indicated that nanoemulsion-based hydrogels were safer and less irritating than free oil-based hydrogels. Enhanced skin penetration was confirmed through confocal microscopy, showing that nanoemulsion-based hydrogels enabled deeper delivery of active ingredients compared to free oil formulations.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig1: SEM image of (A) TTO crosslinked hydrogel, SEM image of (B) RMO crosslinked hydrogel. Arrows indicate nanoemulsion within the hydrogel matrix
Fig 2; Skin irritation test results after applying hydrogel formulations at T=0, after 24 hrs and 72hr
Fig 3: Ex-vivo skin permeation profile TTO loaded nanoemulsion based hydrogel formulation
Fig 4: Ex-vivo skin permeation profile RMO loaded nanoemulsion based hydrogel formulation
Fig 5: CLSM of A- Rhodamine loaded nanoemulsion-based hydrogel (TH3/RH3) CLSM of B- Rhodamine loaded free oil (TTO6/RMO6)
Fig 6: Comparison of Nitric oxide scavenging capacity of oil & hydrogel formulation
Fig 7: Comparison of DPPH radical scavenging capacity of oil & hydrogel formulation
Fig 8: Comparison of SPF values of Essential oils, Base hydrogel and different hydrogel formulations.
Fig 9: Zone of Inhibition graph of antibacterial effect of TH3 formulation
Fig 10; Zone of Inhibition graph of antibacterial effect of RH3 formulation
DETAILED DESCRIPTION OF THE INVENTION
The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.
Tea tree /Rosemary was used as an active component for the formulation of nanoemulsion for age-defying and antimicrobial activity. Tween 80, propylene glycol was used as Smix for the nanoemulsion preparation. Excipients such as oils, surfactants, and co-surfactants play an important role in the formulation of therapeutically effective and stable nanoemulsion formulation.

The present invention relates to a nanoemulsion-based hydrogel formulation for age-defying and antimicrobial activity using essential oils.

Generally, in the present invention the nanoemulsion (NEs) can be made using a variety of techniques. To summarize, the drug is to be dissolved in oil with a surfactant and a co-surfactant, and then slowly mixed with water while stirring until the system is clear. Pseudoternary phase diagrams are used to calculate the percentage of oil phase, volume of surfactant, co-surfactant, and water to be applied. Results from the pseudo ternary phase diagram, shows that the nanoemulsion prepared using 1:1 and Smix: oil ratio 6:4 weight ratios of TTO/Propylene glycol/Tween 80 and RMO/Propylene glycol/Tween were selected for further studies. The composition of the developed nanoemulsion based on essential oil selected according to low, medium, and high (-1, 0, +1) concentration of oil for further characterization
EXAMPLE 1: COMPOSITION OF NANOEMULSION
By trial and error method, the concentration range of oil and Smix was determined based on water uptake in the formulation and the percentage transparency which was found to be 2.5-10% w/w for oil and 50-60% w/w for Smix. The %w/w of oil and Smix was optimized based on the consideration that a high concentration of surfactant could cause toxicity and skin irritation. 60 %w/w Surfactant/Co-surfactant was selected as the maximum safe concentration. 47.5% w/w water proportion was selected. Further, 37.5%w/ w of surfactant and 12.5% w/w of co-surfactant were selected as minimum requirements for stable and successful nanoemulsion formulation, and 10%w/w of oil was selected as the highest quantity of oil that can be incorporated to form a stable nanoemulsion.

EXAMPLE 2: Characterization of Nanoemulsions
Optical Microscopy
To optimize the optical microscopy of the formulation, the sample solution will be placed in a clean, transparent container under high-quality illumination to prevent light scattering into the eyes. It will be viewed against a dark background with a black-and-white reflection to enhance contrast and improve the visibility of the sample. Results shows that All of the nanoemulsions were determined to be clear and transparent, when viewed against dark and white backgrounds.
Determination of pH
The pH of the solution will be measured using a pH meter equipped with a glass electrode. This measurement reflects the hydrogen ion concentration in the sample, as represented by the pH value.
Results shows that the pH values observed for optimized rosemary oil-loaded nanoemulsion (RM6) was 4.69±0.03 & TTO oil-loaded nanoemulsion (TM6) was 5.59±0.04.
Results from the viscosity studies shows that for optimized rosemary oil-loaded nanoemulsion (RM6) 1.844±0.010 & TTO oil-loaded nanoemulsion (TM6) 1.993±0.019.
Results from the Phase separation studies shows that the phase separation of TTO/ RMO loaded nanoemulsion formulation on 30min time interval was found to be 6.88±0.34 & 6.88±0.34 respectively
Freeze-Thaw Cycle (FTC)
The freeze-thaw cycle (FTC) involves temperature fluctuations that cause water to freeze and subsequently thaw as temperatures pass above and below the freezing point (32°F). FTCs are often most common in winter but can occur year-round. In general, FTC is calculated by taking the total number of times temperatures cross the 32°F threshold within a period and dividing by two.
Table 1: Freeze-thaw cycle for Tea tree oil-loaded nanoemulsion
Batches Freeze-thaw cycle freezing at (-18ºc for 20 hrs) Observation Thaw cycle to oven at (40 ºc for 2 hrs) Observation
TM1 After 20 hrs. No change After 2 hrs. Change
TM2 After 20 hrs. No change After 2 hrs. No change
TM3 After 20 hrs. Change After 2 hrs. No change
TM4 After 20 hrs. Change After 2 hrs. Change
TM5 After 20 hrs. No change After 2 hrs. No change
TM6 After 20 hrs. No Change After 2 hrs. No Change
TM7 After 20 hrs. No change After 2 hrs. No change
TM8 After 20 hrs. Change After 2 hrs. Change
TM9 After 20 hrs. Change After 2 hrs. No change

Table 2: Freeze-thaw cycle for Rosemary oil-loaded nanoemulsion
Batches Freeze-thaw cycle freezing at (-18ºc for 20 hrs) Observation Thaw cycle to oven at (40 ºc for 2 hrs) Observation
RM1 After 20 hrs. No change After 2 hrs. No change
RM2 After 20 hrs. No change After 2 hrs. No change
RM3 After 20 hrs. No change After 2 hrs. No change
RM4 After 20 hrs. No change After 2 hrs. Change
RM5 After 20 hrs. No change After 2 hrs. Change
RM6 After 20 hrs. No change After 2 hrs. No Change
RM7 After 20 hrs. No change After 2 hrs. No Change
RM8 After 20 hrs. No change After 2 hrs. Change
RM9 After 20 hrs. No change After 2 hrs. Change
Encapsulation Efficiency
1, 8-cineole one of the major components of TTO/RMO, was chosen as an index for the determination of encapsulation efficacy. The nanoemulsion was placed in a centrifuge tube and subjected to centrifugation at 30,000 rpm for one hour at 4°C using a cooling centrifuge. Following centrifugation, the supernatant and sediment were separated, and their volumes were recorded. The supernatant was lysed using an acetonitrile-water solution (85:15) and filtered. High-performance liquid chromatography (HPLC) was used to assay the essential oil in both the supernatant and sediment. Encapsulation efficiency was then calculated using the formula:
% Encapsulation Efficiency = ((Ct - Cs) / Ct) × 100,
where Ct is the total essential oil in both supernatant and sediment, and Cs is the essential oil in the supernatant.
Zeta Potential and Size Distribution Measurement
The mean particle size and zeta potential of the essential oil-loaded nanoemulsion were measured using a dynamic light scattering technique. To measure globule size, the nanoemulsion was diluted with distilled water and placed in the Zetasizer cuvette. Measurements were conducted at 25°C, with data collected on globule size and distribution.
Table 1: Composition of different batches of RMO-loaded nanoemulsion preparation
Batches % Oil % Smix Globule size (nm) ±SD % Transmittance Zeta potential (mv) PDI
RM1 2 50 55.49±0.06 77.6 -7.8±0.06 0.16± 0.2
RM2 4 50 162.50±0.12 79.3 1.08±0.26 0.14± 0.3
RM3 6 50 196.80±0.56 81.9 2.36±0.86 0.07± 0.4
RM4 2 55 19.56±0.78 80.6 -13.8±0.83 0.08± 0.1
RM5 4 55 176.5±0.12 91.8 2.66±0.23 0.06± 0.0
RM6 6 55 18.09±0.06 96.7 -5.9±0.02 0.67± 0.1
RM7 2 60 16.15±0.23 87.6 -16.8±0.23 0.11± 0.2
RM8 4 60 95.77±0.03 82.6 -0.98±0.63 0.10± 0.0
RM9 6 60 157.4±0.13 81.9 2.98±0.32 0.18± 0.3

Table 2: Composition of different batches of TTO-loaded Nanoemulsion preparation
Batches % Oil % Smix Globule size (nm) ±SD % Transmittance Zeta potential (mv) PDI
TM1 2 50 56.44±0.01 82.6 -7.63±0.12 0.14± 0.2
TM2 4 50 169.8±0.03 86.4 3.93±0.02 0.12± 0.3
TM3 6 50 198.9±0.03 77.9 2.56±0.04 0.07± 0.4
TM4 2 55 19.56±0.12 81.9 -12.89±0.03 0.09± 0.1
TM5 4 55 179±0.123 90.8 2.89±0.012 0.08± 0.0
TM6 6 55 18.67±0.03 95.9 -5.43±0.03 0.98± 0.1
TM7 2 60 16.29±0.02 85.6 16.75±0.04 0.12± 0.2
TM8 4 60 94.88±0.01 91.6 -0.06±0.02 0.11± 0.0
TM9 6 60 157.9±0.01 80.6 2.93±0.03 0.19± 0.3
From the results optimization of a final batch of the nanoemulsion, the 6th batch (TM6) and (RM6) containing 6% oil and Smix were selected as optimized nanoemulsion, and further characterization tests were done by using batch formulation (TM6) and (RM6).

EXAMPLE 3:
Formation of ADA/ Chitosan Hydrogel
Chitosan (CS) solution (2.5mL, weight = 4% w/v) was produced with 1% w/v ascorbic acid. ADA solution (2.5 mL, weight = 20% w/v) was made with distilled water and combined with the Chitosam solution. The Chitosan-Alginate aldehyde hydrogel was formed by mixing the material on a heated magnetic stirrer at 40-50ºC for 10 minutes. After that, CaCl2 (0.10g) was dissolved in distilled water (10 mL), and 2 mL of this solution was carefully added to the CS/ADA combination, generating the cross-linked hydrogel.

Determination of Gelation Time
A mixture of chitosan and alginate dialdehyde solution treated with calcium chloride solution was transferred to a petri dish, and a magnetic stirrer was placed in the center and stirred at 300 rpm using a hot plate under controlled temperature. The gelation time was recorded when the solution formed a solid globule and separated from the bottom of the dish.

Method of preparation chitosan / ADA crosslinked hydrogel bearing nanoemulsion
The nanoemulsion-based hydrogel was formed by (2, 4, and 6%w/v) the final concentration was selected based on optimized formulation. Then mixing a chitosan solution with an alginate dialdehyde 20% w/v solution using a hot plate and stirring at a controlled temperature of 40-50°C for 10 minutes to form a preliminary hydrogel mixture; adding andrographolide (0.25% w/w) to the preliminary hydrogel mixture, along with either: a tea tree oil-loaded nanoemulsion or a rosemary oil (RMO)-loaded nanoemulsion at a concentration of 2%, 4%, or 6% w/w, or free tea tree oil or free rosemary oil at a concentration of 6% w/w;introducing a calcium chloride (CaCl2) solution to the mixture with stirring at 300 rpm at 37°C for 10 minutes to initiate crosslinking of the hydrogel; continuously stirring the mixture until a stable, crosslinked nanoemulsion-based chitosan/alginate dialdehyde hydrogel formulation is formed.

Table 4: Composition of nanoemulsion-based chitosan / ADA crosslinked hydrogel formulation (% w/w)
Component
(%w/w)
Base Hydrogel TH1 TH2
TH3 TTO6 RH1
RH2 RH3 RMO6
Alginate Dialdehyde (ADA) 20 20 20 20 20 20 20 20 20
CaCl2 1 1 1 1 1 1 1 1 1
Glycerin 2 2 2 2 2 2 2 2 2
Water 35 35 35 35 35 35 35 35 35
Chitosan 4 4 4 4 4 4 4 4 4
andrographolide 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25
TTO loaded in nanoemulsion
….
2
4
6
….
….
….
….
….
RMO loaded in nanoemulsion
….
….
….
….
….
2
4
6
….
Free TTO …. …. …. …. 6 …. …. …. ….
Free RMO …. …. …. …. …. …. …. 6
TH- Tea tree-loaded nanoemulsion-based hydrogel
RH- Rosemary-loaded nanoemulsion-based hydrogel

EXAMPLE 4: Physical Characterization
All prepared hydrogel formulations were evaluated for physical properties, including color, odor, and grittiness. These characteristics were assessed through simple visual observation to ensure the formulation's appearance and texture met the expected standards.
Organoleptic Characteristics Results
Color White
Odor Characteristics mild odour of oil
Phase -separation No
Washability Washable
pH Determination
The pH of the hydrogel formulations was measured by diluting the hydrogel with distilled water at a 1:10 ratio. After thorough mixing, the pH was measured in triplicate using a Digital pH meter.
Results shows that the pH values of all developed formulations were in the range of 6.8±0.052gm.mm/sec to 7.2±0.050 gm. mm/sec.

Viscosity Measurement
The viscosity of the hydrogel was measured at a shear rate of 10.00 s?¹ at a temperature of 25 ± 1°C using a Brookfield viscometer (with a T-spindle (S-93) operating at 20 rpm. This measurement was repeated three times to ensure consistency, and the mean viscosity values were recorded.
The viscosity result of the prepared hydrogel formulations was in the range of 2713.6±1.03cp to 6011.2±1.20cp. The essential oil content determination results indicated that the essential oil was uniformly distributed throughout the nanoemulsion-based hydrogel formulation. It was interesting to observe that in the case of free oil-loaded hydrogel, the oil content results were significantly reduced; this might be due to the loss of free essential oil during the hydrogel formulation process.
Determination of Percent Essential Oil Content
The percentage of essential oil in the hydrogel formulation was determined by diluting 10 mg of the hydrogel with 10 ml of an acetonitrile-water mixture (85:15) and mixing for 40 minutes using a Vortex shaker. The solution was then analyzed by HPLC to measure the oil content accurately.
Table 5: pH, Viscosity, and Percent Essential Oil content of hydrogel formulation
Formulation code pH Viscosity (cp) EO Content (%)
Base Hydrogel 6.8±0.053 2713.6±1.03 …..
TH1 7.2±0.556 5413.8±1.56 90.46±1.96
TH2 6.9±0.596 5402.9±1.58 93.58±1.06
TH3 6.8±0.058 6011.5±1.20 98.23±1.89
RH1 7.1±0.089 5426.5±2.60 95.62±2.01
RH2 7.0±0.453 4413.2±1.89 97.48±2.01
RH3 7.1±0.028 4305.1±1.86 98.55±1.56
TTO6 7.0±0.561 2878.5±1.04 74.78±2.20
RMO6 6.9±0.145 4029.3±1.06 71.86±1.86
Homogeneity, Grittiness, and Spreadability
The homogeneity of the hydrogel formulation was tested by passing a small amount of hydrogel between the thumb and index finger to assess the texture for any coarse particles. Further, spreadability was measured using an apparatus with two glass slides (7.5 mm x 7.5 mm), a wooden block, and a pulley system. About 1 gram of the hydrogel was placed between the slides, a 100-gram weight was applied, and then a 30-gram weight pulled the upper slide. The time taken for the slide to move a 5-mm distance was recorded, with shorter times indicating better spreadability.
The spreadability result of prepared hydrogel was found in the range of 17.69± 0.23gm.mm/sec to 25.50±0.34gm.mm/sec. The result showed that prepared hydrogel can spread by a small amount of shear.
Table 6: Homogeneity, Grittiness and Spreadability
Formulation code Homogeneity Grittiness Spreadability (gm.mm/sec)
Base Hydrogel Good No 17.69±0.45
TH1 Good No 21.89±0.89
TH2 Good No 22.45±0.56
TH3 Good No 25.86±0.45
RH1 Good No 18.06±0.78
RH2 Good No 20.78±0.23
RH3 Good No 22.45±0.14
TTO6 Good No 20.60±0.78
RMO6 Good No 18.63±0.49

Stability Studies of Hydrogel Formulation
Stability testing was conducted to verify the hydrogel formulation's chemical, physical, and aesthetic properties under various storage conditions: 8°C ± 2°C, room temperature (25°C), and 40°C ± 2°C. Over a 30-day period, the formulation was monitored for color, texture, phase separation, pH, viscosity, spreadability, and active content.
Table 7: Results for pH and viscosity of hydrogels after 30 Days
Formulation code pH Viscosity (cp)
08ºC RT 40ºC 08ºC RT 40ºC
TH1 6.9±0.01 7.0±0.02 6.0±0.03 2524.5±0.01 2613.1±1.02 2078.3±1.22

TH2 6.8±0.02 6.5±0.04 6.4±0.04 5220.5±1.56 5420.2±1.06 5068.8±1.46

TH3 6.8±0.03 6.6±0.02 6.0±0.03 5040.1±1.20 5280.2±1.04 4824.4±1.40

RH1 6.9±0.01 6.5±0.04 6.0±0.01 5961.4±1.23 6061.0±1.07 5410.2±1.24

RH2 7.0±0.04 7.0±0.02 6.2±0.03 5241.3±1.20 5342.1±1.03 5027.5±1.24

RH3 7.0±0.03 6.9±0.03 6.8±0.01 4002.1±1.01 4545.2±1.00 4125.6±1.2
Table 8: Results for Spreadability and Percent Oil Content after 30 Days
Formulation code (%) Oil Content Spreadability (gm. mm/sec)
08ºC RT 40ºC 08ºC RT 40ºC
TH1 89.9±0.01 89.0±0.02 88.0±0.03 18.95±0.01 13.1±1.02 25.23±1.22

TH2 90.8±0.02 93.5±0.04 86.4±0.04 18.85±1.56 20.2±1.06 30.08±1.46

TH3 96.8±0.03 98.6±0.02 92.0±0.03 20.46±1.20 10.2±1.04 29.04±1.40

RH1 90.9±0.01 92.5±0.04 90.0±0.01 15.14±1.23 22.0±1.07 26.21±1.24

RH2 95.0±0.04 95.0±0.02 95.2±0.03 17.36±1.20 18.1±1.03 25.15±1.24

RH3 97.0±0.03 97.9±0.03 92.8±0.01 20.68±1.01 15.2±1.00 28.16±1.20


SEM Analysis (Scanning Electron Microscopy)
The surface morphology of the chitosan/ADA crosslinked hydrogel was observed using a scanning electron microscope. A drop of 1% aqueous phosphotungstic acid solution was applied to the sample for five minutes to enhance visibility, followed by drying. The hydrogel was then examined under SEM with an acceleration voltage of 80 kV. Figure 1 displays the SEM image of (A) TTO crosslinked hydrogel, SEM image of (B) RMO crosslinked hydrogel. Arrows indicate nanoemulsion within the hydrogel matrix
Skin Irritation Study
A skin irritation study was conducted using albino rats, divided into control and test groups. After shaving a marked area (5 mm²) on each rat, the hydrogel formulation was applied, and skin reactions were monitored for three days using the Draize scoring system. Erythema and edema scores at 24, 48, and 72 hours were recorded to calculate the Primary Irritation Index (PII), which categorized irritation severity as negligible, slight, moderate, or severe. Figure 2 displays the Skin irritation test results after applying hydrogel formulations at T=0, after 24 hrs and 72hrs.
Table9: Scores for Skin Irritation Study
Skin Reaction Time (hrs) Group 1
(Hydrogel base) Group 2
(Formulation TH3) Group 3
(Formulation RH3)
Rat-1 Rat-2 Rat-3 Rat-1 Rat-2 Rat-3 Rat-1 Rat-2 Rat-3

Erythema 24 1 1 1 1 1 1 1 0 0
48 0 0 0 0 0 0 0 0 0
72 0 0 0 0 0 0 0 0 0

Edema 24 0 0 0 1 0 1 1 1 0
48 0 0 0 0 0 0 0 0 0
72 0 0 0 0 0 0 0 0 0
The score of Primary Irritation 0.16 0.16 0.16 0.33 0.16 0.33 0.33 0.16 0.0
Primary Irritation Index 0.16 0.27 0.16
Skin Reaction Time (hrs) Group 4
(TTO6) Group 5
(RMO6)
Rat-1 Rat-2 Rat-3 Rat-1 Rat-2 Rat-3

Erythema 24 1 1 1 1 1 1
48 1 0 1 1 1 1
72 0 0 0 0 0 0

Edema 24 1 1 1 1 1 1
48 1 0 1 0 1 0
72 0 0 0 0 0 0
The score of Primary Irritation 0.66 0.33 0.66 0.50 0.66 0.33
Primary Irritation Index 0.55 0.49
The results obtained from the primary skin irritation studies are listed in Table 5.28, and interpreted according to the Draize test which says that test samples that produce PII scores of 2 or less are considered negative i.e. no skin irritation. Since the score between 0 and 2 suggests no to mild irritation, the low PII of Nanoemulsion-based hydrogel formulation (0.27 for TTO-loaded hydrogel and 0.16 for RMO-loaded Nanoemulsion based hydrogel formulation) in comparison to base hydrogel (0.16) observed in the study depicted non- irritancy of the hydrogel formulation and could be considered as safe for use.

The primary skin irritation studies of free oil-loaded hydrogel formulation showed high PII values (0.55 for TTO-loaded hydrogel and 0.49 for RMO-loaded hydrogel formulation) indicating slight skin irritation in comparison to nanoemulsion-based hydrogel formulation of the same oil.
Ex Vivo Skin Permeation Studies
Ex vivo permeation studies were performed using Franz diffusion cells with an effective diffusion area of 2 mm². The hydrogel was applied to the donor compartment, and the receptor compartment, filled with PBS (pH 5.4) at 37 ± 1°C, was stirred at 100 rpm. Samples were taken at 30-minute intervals over 8 to 48 hours, filtered, and analyzed by UV spectrophotometry. Drug flux and permeability coefficient (Kp) were calculated to assess the drug's permeation, while the enhancement ratio (ER) was determined by comparing flux values with a control.
Various permeability parameters such as Jss, Kp, and ER were significantly enhanced in the nanoemulsion-based hydrogel formulation compared to the free oil-loaded hydrogel formulation. Enhanced Ex-vivo permeation results show that the formulation TH3/RH3 (Containing oil 6%) was considered an optimized formulation. Fig 3& 4 clearly shows that the Ex-vivo skin permeation profile TTO loaded nanoemulsion based hydrogel formulation, RMO loaded nanoemulsion based hydrogel formulation, respectively.

Table 10: Ex-vivo permeability parameter of prepared hydrogel formulation

Formulation code Jss±SD
(mg/mm2/h) Kp±SD
(mm/hx10-3) ER
Base Hydrogel 0.0689 ±0.002 1.456 ±0.078 -
TH1 0.1765 ±0.022 3.562 ±0.045 2.36
TH2 0.2156 ±0.025 4.856 ±0.036 3.07
TH3 0.2566 ±0.089 5.189 ±0.078 3.58
RH1 0.1789 ±0.096 3.568 ±0.063 2.66
RH2 0.2188 ±0.056 4.889 ±0.014 3.18
RH3 0.2655 ±0.023 5.896 ±0.075 3.79
TTO6 0.1788 ±0.078 2.569 ±0.023 1.89
RMO6 0.1899 ±0.063 2.599 ±0.078 1.98
(Mean ± SD, n=3)

EXAMPLE 5; Confocal Laser Scanning Microscopy (CLSM) Study
Nanoemulsion-based Hydrogel formulations loaded with TTO/RMO essential oil were prepared, and penetration across the skin was measured by CLSM study. Skin permeation of rhodamine (marker) from rhodamine-loaded nanoemulsion-based hydrogel (TH3 & RH3) and rhodamine-loaded free oil hydrogel (TTO6 & RMO6) was visualized through a confocal laser scanning microscope. It was observed that permeation from rhodamine-loaded free oil hydrogel (Figure B) was confined only to the upper layer of the skin epidermis, while in the case of rhodamine-loaded nanoemulsion-based hydrogel (Figure A), enhanced permeation of rhodamine was observed deep into the skin layers. This shows that prepared nanoemulsion-based hydrogel has the ability to carry antiaging EO deep into the skin layers. Figure 5 clearly shows that the CLSM of A- Rhodamine loaded nanoemulsion-based hydrogel (TH3/RH3) CLSM of B- Rhodamine loaded free oil (TTO6/RMO6)

EXAMPLE 6: Antioxidant Capacity Assessment
The antioxidant capacity was assessed for free essential oils (Rosemary Oil - RMO and Tea Tree Oil - TTO), TTO/RMO-based nanoemulsion formulations, and free oil-loaded hydrogel formulations containing 6% oil.
Nitric Oxide Scavenging Activity
This activity measures the antioxidant's ability to neutralize nitric oxide by reducing nitrite ion production. Standard ascorbic acid showed the highest scavenging activity at 250 µg/ml (92.66% ± 0.01), while TTO and RMO demonstrated 81.57% ± 0.34 and 90.04% ± 0.23 scavenging capacities, respectively, with IC50 values of 18.01 µg/ml (TTO) and 26.84 µg/ml (RMO).

Nanoemulsion-Based Hydrogel vs. Free Oil Hydrogel
In TTO-loaded hydrogels, the scavenging capacity ranged from 61.03% to 81.74% with IC50 values from 2.18 µg/ml to 98.59 µg/ml. For RMO-loaded hydrogels, the capacity ranged from 59.86% to 76.23% with IC50 values between 21.87 µg/ml and 159.41 µg/ml. Nanoemulsion-based hydrogels generally exhibited higher scavenging capacities compared to free oil-loaded hydrogels, indicating that nanoemulsion formulations enhance the antioxidant stability and efficacy of the essential oils. This improved performance in nanoemulsion-based hydrogels may be due to reduced volatility and better encapsulation of the oils, limiting loss and instability observed in free oil formulations.. Figure 6 displays the comparison of nitric oxide scavenging activities across free oil, base, nanoemulsion-based, and free oil hydrogel formulations.

Table 11:DPPH scavenging assay
Code Ascorbic acid TTO RMO Base hydrogel
% Inhibition (at250 µg/ml) 96.14±0.01 83.34±0.46 88.44±0.36 61.88±0.11
IC50 (µg/ml) 10.39 18.68 29.69 92.66
Code TH1 TH2 TH3 TTO6
% Inhibition
(at 250 µg/ml) 64.92±0.13 77.64±0.34% 88.96±0.36 78.65±0.45
IC50 (µg/ml) 91.87 47.88 10.13 48.76
Code RH1 RH2 RH3 RMO6
% Inhibition
(at 250 µg/ml) 65.89±1.09 70.56±2.99 82.96±1.06 66.56±1.98
IC50 (µg/ml) 157.12 72.12 25.38 87.11
Figure 7 shows the Comparison of DPPH scavenging capacity capacity of oil & hydrogel formulation
EXAMPLE 7: Determination of In-vitro Sun Protection Factor
In-vitro SPF determination is a useful test for screening ingredients during the development stage of a cosmeceutical product.

The higher the SPF, the more protection is offered by a sunscreen against UV light. An essential oil should absorb major UV radiations (290-400nm) to be adequately used in cosmeceutical formulations to prevent photoaging, sunburn, skin wrinkles, and other skin damage. In the present investigation, the SPF value of TTO and RMO was found to be table 12 respectively.
Essential oil-loaded nanoemulsion formulation showed remarkably high SPF in comparison to essential oil alone and base hydrogel. The SPF value of TTO-loaded nanoemulsion hydrogel formulation was found to be 6.04, 8.25, and 10.33 for TH1, TH2, and TH3 respectively (Table 13). The SPF of RMO-loaded nanoemulsion-based hydrogel formulation was found to be 9.11, 12.28, and 18.65 for RH1, RH2, and RH3 respectively (Table 14). The SPF of free oil-loaded hydrogel formulation was 7.86 (TTO loaded) and 9.18 (RMO loaded) which is very low in comparison to the Nanoemulsion hydrogel at the same concentration of essential. Fig 8 shows the comparison of SPF values.
Table 12: SPF (Sun Protection Factor) Value of TTO/RMO and Base hydrogel
Wavelength ( nm) Absorbance of
TTO Absorbance of RMO Base Hydrogel
290 0.905± 0.08 0.955± 0.14 0.611±0.09
295 00867± 0.11 0.939± 0.08 0.589±0.06
300 0.785± 0.07 0.899± 0.16 0.445±0.10
305 0.630± 0.16 0.830± 0.19 0.278±0.13
310 0.523± 0.09 0.799± 0.18 0.235±0.05
315 0.445± 0.07 0.732± 0.12 0.198±0.09
320 0.433± 0.11 0.675± 0.14 0.168±0.09
Calculated Sun Protection Factor 6.85 8.45
3.37
Table 13: SPF (Sun Protection Factor) Value of TTO loaded nanoemulsion based hydrogel formulation
Wavelength (nm) Absorbance
TH1 TH2 TH3 TTO6
290 0.189± 0.78 0.874± 0.01 1.012±0.09 0.015±0.02
295 0.333± 0.13 0.709± 0.02 1.889±0.06 0.083±0.13
300 0.485± 0.06 0.878± 0.01 0.945±0.30 0.289±0.12
305 0.625± 0.01 0.785± 0.11 0.978±0.03 0.325±0.11
310 0.813± 0.01 0.899± 0.01 0.895±0.05 0.186±0.11
315 0.745± 0.02 0.932± 0.12 0.878±0.01 0.089±0.09
320 0.538± 0.01 0.975± 0.02 0.668±0.02 0.018±0.10
Calculated Sun Protection Factor 6.04 8.25
10.33
7.86
TABLE 14: SPF (Sun Protection Factor) Value of RMO loaded nanoemulsion based hydrogel formulation
Wavelength ( nm) Absorbance
RH1 RH2 RH3 RMO6
290 1.189± 0.01 1.974± 0.01 1.912±0.09 0.785±0.02
295 1.803± 0.14 1.709± 0.02 1.889±0.06 0.883±0.13
300 0.785± 0.05 1.978± 0.01 2.945±0.30 0.989±0.12
305 0.801± 0.02 0.885± 0.11 1.678±0.03 0.725±0.11
310 0.919± 0.01 0.899± 0.01 0.995±0.05 0.986±0.11
315 0.998± 0.02 0.992± 0.12 0.978±0.01 0.889±0.09
320 0.986± 0.01 0.685± 0.02 0.868±0.02 0.858±0.10
Calculated Sun Protection Factor 9.33 12.28
18.65
9.18
In-vitro Enzyme Inhibition Assay
Two optimized formulations from each essential oil were used for the further investigation of the enzyme inhibition assay. From the results of the collagenase inhibition assay, it was observed that both the formulations showed anti-collagenase activity in a concentration-dependent manner. It was observed that the anti-collagenase activity increases as the concentration increases from 1-100 µg/ml. In the case of TH3 formulation maximum inhibition (27.8%) was observed at 1000 µg/ml, whereas at the same concentration, RH3 showed the maximum inhibition of 23.9%.
In-Vitro Elastase Inhibition Assay
The anti-elastase activities of both the optimized hydrogel formulations (TH3 & RH3). The anti-elastase activity was found to be in a concentration-dependent manner. In the case of TTO-loaded nanoemulsion hydrogel formulation (TH3), it was surprising to see that the antielastase inhibition was higher (144.0%) than EGCG (123.4%) at the concentration of 1000 µg/ml. RMO-loaded nanoemulsion-based hydrogel formulation (RH3) also showed significant elastase inhibition activity. The highest anti-elastase inhibition was observed at 109.2% at the same concentration i.e. 1000 µg/ml.
In-vitro Antibacterial activity
Determination of Antibacterial activity for TH3 (Optimized) Formulation
The antibacterial activity of optimized nanoemulsion-based hydrogel formulation (TH3/RH3) and optimized free oil-loaded hydrogel formulation (TTO6/RMO6) has been determined by agar disc diffusion assay method. Streptomycin was used as the standard marketed formulation, It was shown that the highest zone of inhibition against the gram (+) Staphylococcus aureus and gram (-) bacteria Escherichia coli, zone of inhibition of TH3/RH3 shown in Table 15.
Table: 15: Zone of Inhibition of Optimized hydrogel (TH3) and Standard formulation
Bacterial strains


Staphylococcus aureus
(gram-positive) Formulation code Zone of Inhibition
Marketed formulation (streptomycin) 4.6±0.1mm
Optimized nanoemulsion based hydrogel formulation (TH3) 4.8±0.2mm
Free oil-loaded hydrogel formulation (TTO6) 1.9±0.1mm
Tea tree Essential oil 1.1 ±0.2mm
Base Hydrogel 0.9±0.2mm


Escherichia coli
(gram-negative) Marketed formulation (streptomycin) 4.3 ±0.1mm
Optimized nanoemulsion based hydrogel formulation (TH3) 2.1±0.2mm
Free oil-loaded hydrogel formulation (TTO6) 1.6±0.2mm
Tea tree Essential oil 1.3 ±0.1mm
Base Hydrogel 1.0±0.1mm
Determination of Antibacterial activity for RH3 (Optimized) Formulation
The zone of inhibition range has been measured for marketed formulation at 4.3mm and Optimized Nanoemulsion hydrogel formulation (RH3) at 3.4mm in Staphylococcus aureus. Whereas the same formulation on gram (-) bacteria Escherichia coli has been shown 4.1mm and 2.0mm for Standard formulation & RH3 respectively.

Table 16: Zone of Inhibition of Optimized Hydrogel (RH3) and Standard Formulation

Bacterial strains
Formulation code Zone of Inhibition


Staphylococcus aureus
(gram-positive) Marketed formulation (streptomycin) 4.3±0.1mm
Optimized nanoemulsion based hydrogel formulation (RH3) 3.4±0.2mm
Free oil-loaded hydrogel formulation (RMO6) 1.6±0.1mm
Rosemary Essential oil 1.1±0.2mm
Base Hydrogel 0.8±0.1mm


Escherichia coli
(gram-negative) Marketed formulation (streptomycin) 4.1 ±0.2mm
Optimized nanoemulsion based hydrogel formulation (RH3) 2.0±0.1mm
Free oil-loaded hydrogel formulation (RMO6) 1.5±0.2mm
Rosemary Essential oil 0.9±0.1mm
Base Hydrogel 0.6±0.1mm
Determination of antifungal activity
The zone of inhibition range has been shown for Optimized nanoemulsion-based hydrogel formulation (TH3) 3.7±0.1mm and the marketed formulation shows 4.5 ±0.1mm. The result shows that TH3 higher zone of inhibition as compared with RH3 hydrogel formulation. The zone of inhibition range shown for optimized hydrogel formulation was 2.1±0.1mm (RH3).

Table: 17 Zone of Inhibition of Optimized Hydrogel (TH3/RH3) and Standard Formulation
Fungal Strains
Formulation code Zone of Inhibition




Candida albicans Marketed formulation (Clotrimazole) 4.5±0.1mm
Optimized nanoemulsion based hydrogel formulation (TH3) 3.7±0.1mm
Free oil-loaded hydrogel formulation (TTO6) 1.8±0.1mm
Base Hydrogel 0.6±0.1mm
Tea tree essential oil 1.4±0.1mm
Marketed formulation (Clotrimazole) 4.1±0.1mm
Optimized nanoemulsion based hydrogel formulation (RH3) 3.6±0.1mm
Free oil-loaded hydrogel formulation (RMO6) 1.9±0.1mm
Base Hydrogel 0.9±0.1mm
Rosemary essential oil 1.6±0.1mm


Effect of TH3 formulation on various parameters of (+) Gram & (-) Gram bacteria and Candida albicans
Table: 18 The effect of TH3 formulation on various physiological parameters
Bacteria Physiological parameters of microorganisms
Decrease cell growth
[%] ?Propidium iodide staining (PI) [%] Absorbing material leakage at 280 nm
(%) Potassium ion effect
(%) Decrease the respiration rare
(%)
Escheriahia
Coli 89.0 84.0 17.0 81.0 80.0
Staphylococcus
Aureus 27.0 12.0 14.0 18.0 26.0
Candida
Albicans 73.0 60.0 10.0 16.0 68.0
Effect of RH3 formulation on various parameters of (+) Gram & (-) Garm bacteria and Candida albicans
Table: 19 The effect of RH3 formulation on various physiological parameters



Bacteria Physiological parameters of microorganisms
Decrease cell growth
[%] ? Propidium iodide staining
[%] Absorbing material leakage at 280nm
(%) Potassium ion effects
(%) Decrease the rate of respiration
(%)
Escheriahia Coli 83.0 79.0 12.0 80.0 75.0

Staphylococcus aureus 29.0 10.0 11.0 15.0 21.0

Candida albicans 69.0 55.0 8.0 14.0 61.0

EFFICACY DATA:
Formulation Code Anti- Oxidant Activity Sun- protection Factor Anti- Bacterial Activity Anti- Fungal Activity
Andrographolide(Alone) 55.8 5.2 2.5 1.8
TH3 88.9 10.3 4.8 3.7
RH3 82.5 18.6 4.3 3.6

While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.
, Claims:We Claim,
1. A crosslinked chitosan/alginate dialdehyde (ADA) hydrogel composition with, anti-aging, antimicrobial and photoprotective properties, comprising:
a) 20% w/w alginate dialdehyde (ADA);
b) 1% w/w calcium chloride (CaCl2);
c) 2% w/w glycerin;
d) 35% w/w water;
e) 4% w/w chitosan;
f) 0.25% w/w andrographolide;
g) an essential oil component selected from tea tree oil or rosemary oil or both;
h) wherein the oil component is incorporated in a nanoemulsion at 2%, 4%, or 6% w/w;
i) free tea tree oil or rosemary oil at 6% w/w for formulations without nanoemulsion;
wherein the hydrogel composition is characterized by:
j) the inclusion of andrographolide and nanoemulsion-based essential oils, providing prolonged and controlled bioavailability with antimicrobial, anti-aging, and photoprotective effects for topical use.
2. The crosslinked chitosan/alginate dialdehyde (ADA) hydrogel composition as claimed in claim 1, wherein the method of preparation of hydrogel comprising the steps of:
a) mixing a chitosan solution with an alginate dialdehyde solution and stirring at a controlled temperature of 40-50°C for 10 minutes to form a preliminary hydrogel mixture;
b) adding andrographolide (0.25% w/w) to the preliminary hydrogel mixture, along with either:
I. a tea tree oil-loaded nanoemulsion or a rosemary oil (RMO)-loaded nanoemulsion at a concentration of 2%, 4%, or 6% w/w, or
II. free tea tree oil or free rosemary oil at a concentration of 6% w/w;
c) introducing a calcium chloride (CaCl2) solution to the mixture with stirring at 300 rpm at 37°C for 10 minutes to initiate crosslinking of the hydrogel;
d) continuously stirring the mixture until a stable, crosslinked nanoemulsion-based chitosan/alginate dialdehyde hydrogel formulation is formed.
3. The crosslinked chitosan/alginate dialdehyde (ADA) hydrogel composition, as claimed in claim 1, wherein the composition of nanoemulsion comprising of:
a) 10% w/w oil, selected from tea tree oil or rosemary oil;
b) 47.5% w/w water, serving as the continuous phase to enable stability and dispersion of nano-sized droplets;
c) 37.5% w/w, Polysorbate 80 (Tween 80) as the surfactant, ensuring nano-sized droplet stability and reducing interfacial tension; and
d) 12.5% w/w propylene glycol as the co-surfactant, enhancing solubility, compatibility with the surfactant, and the overall stability of the nanoemulsion;
e) wherein the selected ratios of oil, water, surfactant, and co-surfactant are optimized to form a nanoemulsion with droplet sizes of less than 200 nm, achieving high stability, low skin irritation, and suitable for controlled delivery
4. The crosslinked chitosan/alginate dialdehyde (ADA) hydrogel composition as claimed in claim 1, wherein the chitosan solution is prepared at a concentration of 4% w/w for optimal hydrogel viscosity and film-forming properties.
5. The crosslinked chitosan/alginate dialdehyde (ADA) hydrogel composition as claimed in claim 1, wherein the tea tree oil-loaded nanoemulsion is prepared using a surfactant-to-co-surfactant ratio of 3:1 to achieve optimal stability and controlled release.
6. The crosslinked chitosan/alginate dialdehyde (ADA) hydrogel composition as claimed in claim 1, wherein the rosemary oil (RMO)-loaded nanoemulsion is prepared with a surfactant-to-co-surfactant ratio of 2:1 to enhance the solubility and stability.
7. The crosslinked chitosan/alginate dialdehyde (ADA) hydrogel composition as claimed in claim 1, wherein the andrographolide is dispersed evenly throughout the hydrogel matrix, providing stability and sustained release for its anti-inflammatory and antimicrobial effects.
8. The crosslinked chitosan/alginate dialdehyde (ADA) hydrogel composition as claimed in claim 1, wherein the hydrogel exhibits antimicrobial activity against common pathogens such as Staphylococcus aureus, Escherichia coli, and Candida albicans.
9. The crosslinked chitosan/alginate dialdehyde (ADA) hydrogel composition as claimed in claim 1, wherein the final hydrogel formulation demonstrates photoprotective effects with a sun protection factor (SPF) value of 6-18.
Dated this 19 November 2024

Dr. Amrish Chandra
Agent of the applicant
IN/PA No: 2959

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