The Formulation, Development And Evaluation of Ointment for Anti-Inflammatory Activity

: The search for safer anti-inflammatory agents has led to exploration of natural sources such as curcumin and fenugreek. Curcumin, derived from Curcuma longa, and α-linolenic acid from Trigonella foenum-graecum, exhibit significant anti-inflammatory properties. This study focuses on formulating an ointment utilizing these extracts for topical application. Molecular docking techniques were employed to assess the binding affinities of curcumin and α-linolenic acid. The ointment was prepared using various bases and preservatives, and its physicochemical properties were evaluated. Stability studies revealed consistent properties under different temperature conditions. Characterization of the drug extracts and ointment formulations was conducted to ensure efficacy and safety. The ointment showed promising results in terms of stability and efficacy, particularly formulation F2, suggesting its potential as a topical anti-inflammatory therapy with improved patient compliance.


INTRODUCTION:
The array of side Effects associated with available Anti-inflammatory drugs (both steroidal and nonsteroidal) has prompted numerous studies to explore natural sources for alternative anti-inflammatory agents.The Curcumin and Fenugreek these are traditionally used as anti-inflammatory Activity.Curcumin demonstrates a wide range of pharmacological effects, including anti-inflammatory, antioxidant, anticancer, antimicrobial, neuroprotective, and cardioprotective properties [1] .Fenugreek exhibit the various pharmacological effects, including hypoglycemic [2] , hypocholesterolemia [3] , antioxidants [4] , and appetite-stimulating properties [5] .Additionally, it demonstrates gastroprotective activity [6] [7] , and histopathological examinations of the liver and brain indicate significant protection against ethanol toxicity [8] with aqueous extracts of fenugreek seeds.Known as "Shanbalileh" in Iranian traditional medicine, this plant has been utilized for its hypoglycemic and antirheumatic properties [7] .this study exhibits he topical preparation of ointment by using curcumin (extracted from Curcuma Longa L. belonging to family Zingiberaceae) and α-Linolenic Acid (extracted from Trigonella foenum-graecum L. belonging to family Fabaceae)

Molecular Docking of Curcumin and α-Linolenic Acid:
Molecular docking is a computational technique used in drug discovery and molecular biology to predict the binding affinity and orientation of a small molecule (ligand) within the active site of a target protein (receptor) [9] .

Methodology:
1. Software Used: Several Software has been used in the Molecular docking such as ChemDraw, ChemSketch 3D, Discover Studios and Pyrax.ChemDraw is used for the preparation of ligands and the receptors.ChemDraw used to modify and prepare ligands for docking studies by adding or removing functional group, adjusting stereochemistry or optimizing molecular conformation.ChemSketch 3D allows the user to convert the 2D structure into the 3D structure.This can help to the understand the spatial arrangement of atoms and functional group.Discovery Studios is used for the preparation of the ligand structure for docking studies.Discovery Studios mainly used in docking algorithm including rigid-body docking, flexible docking and inducing the fit docking.This algorithm allows for the prediction of ligand binding modes and binding affinities, taking into account both ligand and protein flexibility.Pyrax is the python-based molecular docking software that allows users to perform docking simulation and virtual screening studies.The molecular docking calculation is done with the help of Autodock Tools.

Docking Procedure: a. Protein preparation:
Protein preparation is the crucial step in molecular docking to ensure the accurate and reliable docking result Three Dimensional coordinates are retrieved from the Brookhaven Protein Data Bank and prepare the PDB file for the Docking programs.

b. Ligand preparation:
In the Ligand Preparation retrieval of ligand structure, ligand cleaning, Ligand Conformational sampling in which the ligand is explore to its flexibility and account of different binding modes using algorithms or molecular dynamics simulation, Geometric optimization, adding hydrogen atoms and Generating 3D Grids and Maps.Ligand preparation is carried out with the help of ChemDraw and ChemSketch 3D.In ligand Cleaning remove any unwanted atoms or functional group from ligand structure that are not relevant to the docking study.c.Docking Procedure: Docking Procedure is carried out with the help of Pyrax Software and Autodock Tool which include the docking algorithms, scoring function and search space.Perform the docking simulation using defined parameters and input structure [10] .

MATERIAL AND METHOD:
Collection, extraction, formula, ointment preparation, evaluation parameter The Rhizomes of Turmeric (Curcuma longa Linn) of family Zingiberaceae and Seed of Fenugreek (Trigonella Foenum-gracum) of family Fabaceae is collected and authenticated from the botanical laboratory's sources.Make it into the powdered form.

Extraction of Curcumin:
The rhizomes were initially cleaned, washed with deionized water, sliced, and sun-dried for one week, followed by drying again at 50°C in a hot air oven for six hours.Subsequently, the dried rhizomes were cut into small pieces, powdered using an electronic mill.A 10-gm sample was taken into a thimble and placed in a Soxhlet apparatus, where 250 ml of ethanol was added and extracted according to their boiling point for seven hours.After extraction, the dark brown extract was cooled, concentrated by heating, resulting in a crude dried extract with a black-orange color.Each raw sample of turmeric underwent a similar extraction method, and the yield was calculated accordingly [11] .

Extraction of Fenugreek:
The α-Linolenic Acid is fatty acid present in the seed of fenugreek i.e.Trigonella Foenum-gracum.Seed Powder of fenugreek was extracted with the solvent Petroleum Ether (60-80 0 C).Macerate the Seed powder in the petroleum ether for approximately 4 days.After complete maceration filtered it and evaporate it to get the Solid form of extract. [12]rmula: CONTENTS Category Quantity Quantity Quantity Taken in F1 1.Initially, the ointment base was prepared by weighing the appropriate quantity of hard paraffin wax and placing it in a porcelain dish on a water bath.Once the hard paraffin wax melted, the remaining ingredients, including lanolin, Cetostearyl alcohol, and white soft paraffin, were added.2. Next, the dry extract of drugs was incorporated into the ointment base using the levigation method.
The powder was first rubbed with a small quantity of the base to form a concentrated ointment base containing finely divided powder uniformly distributed within it.Subsequently, the concentrated ointment was diluted with the remaining quantity of the base by rubbing it with a spatula.3. Finally, the preservative Methylparaben was added to the formulation [13] .

EVALUATION: 1. Colour and Odour:
The Colour and Odour is determined by the Visual Examination.2. Consistency: Consistency is examined by the observing Smoothness and No Grittiness.3. pH: 2.5grams of each formulation were placed into a dry beaker, followed by the addition of 50 ml of water.The beaker containing the ointments was then heated on a water bath at 60-70°C.The pH of the ointments was measured using a pH meter (pH Tutor, Eutech Instruments).These measurements were conducted in triplicate, and the averages of the three readings were recorded. [14]. Loss on drying: The determination of loss on drying involves placing the ointment in a petri dish on a water bath and drying it at a temperature of 105ºC. [15]ercentage loss on drying = 100 × (Initial Weight -Weight after Drying) / Initial Weight 5. Viscosity: The viscosity was assessed using the CAP-2000 Brookfield viscometer.A clean and dry 250 ml beaker was used to hold the test sample, and its viscosity was determined following the standard operating procedure of the viscometer, employing spindle numbers 1 to 4. Each spindle was utilized to measure the viscosity of the sample at speeds ranging from 0.3 to 60 r.p.m.Additionally, the rheological characteristics were evaluated at 25°C using the Brookfield viscometer [16] .6. Spreadability: Spreadability is assessed by sandwiching the excess sample between two slides, which are then compressed to a uniform thickness by applying a predetermined weight for a specific duration.
The time taken to separate the two slides is measured as an indicator of spreadability, where a shorter separation time indicates better spreadability.Spreadability is calculated using the following formula: S=M × L/T Where, S= Spreadability, M= Weight tide to the upper slide, L= Length of glass slide, T= Time taken to separate the slides [17] 7. Extrudability: In the recent study, the evaluation method for assessing ointment formulation extrudability was based on the percentage of ointment extruded from the tube upon the application of finger pressure.A greater quantity extruded indicated better extrudability.The formulation under investigation was filled into a clean, lacquered aluminum collapsible 5 gm tube with a nasal tip opening of 5 mm.Pressure was applied to the tube using a finger, and the extrudability was determined by measuring the amount of cream extruded through the tip under pressure [18].8. Solubility: The substance is soluble in boiling water and mixes well with ethanol, ether, and chloroform.9. Washability: After applying the ointment to the skin, its wash-off resistance was assessed by examining the ease of removal with water.10.Non-Irritancy Test: The ointment was applied to the skin of human subjects, and its effects were observed.

Stability Studies:
The International Conference on Harmonization (ICH) released the harmonized tripartite guidelines on stability testing of new drug substances and products on October 27, 1993.The physical stability test of the herbal ointment was conducted over four weeks under various temperature conditions, including 4ºC, 25ºC, and 37ºC.The ointment demonstrated physical stability at all tested temperatures [19] .

RESULT AND DISCUSSION:
This study to prepare and evaluate an ointment using dry Curcumin extract longa and Petroleum Ether Extract of Fenugreek.The extract was obtained in dried form.Initially, the dry Curcumin extract longa and Petroleum Ether Extract of Fenugreek was characterized, determining properties such as colour, odour, solubility, and melting point for both reference and standard samples.
For ointment preparation, the levigation method was employed to ensure uniform mixing of the turmeric extract with the base, ensuring stability during storage.The powder was initially mixed with a small quantity of the base to create a concentrated ointment base with evenly distributed powder.Subsequently, the concentrated ointment was diluted with the remaining base using a spatula.The physicochemical properties were meticulously examined, providing accurate results for evaluation parameters including spreadability, viscosity, washability, loss on drying, and others.The stability study of the ointment was conducted by storing the formulation at 4ºC, 25ºC, and 37ºC.. Turmeric and Fenugreek has been valued since ancient times for its numerous medicinal properties, including antibacterial, anti-cancer, anti-inflammatory, and antifungal effects.In this study, an ointment was formulated using various bases such as hard paraffin, Cetostearyl alcohol, lanolin (wool fat), and white soft paraffin, along with preservatives like Methylparaben.From the study it is concluded that the formulation F2, is more stable than others.Curcumin and petroleum ether extract of fenugreek are combined with the appropriate ointment bases and preservatives a better therapy and patient compliance can be attained.