FORMULATION AND EVALUATION OF ANTIMICROBIAL OINTMENTS FROM EUPATORIUM GLANDULOSUM HORT. EX KUNTH

Objective: The present study was undertaken to evaluate the antimicrobial activity of an ointment prepared from alcohol, ethyl acetate, and chloroform extracts of Eupatorium glandulosum Hort. ex Kunth. 
Methods: The dried plant materials of E. glandulosum were individually extracted using alcohol, ethyl acetate, and chloroform by Soxhlet method. These three different types of extracts were used for the preparation of ointment and evaluated for the phytochemicals, pH, homogeneity, spreadability, tube extrudability, stability, and antimicrobial activity. The activity of prepared ointments was evaluated against Escherichia coli and Bacillus subtilis and the in vitro antimicrobial activity of ointments of plant extracts was performed by cup plate methods. The most effective antimicrobial ointment was determined by comparing the results of the zone of inhibition of all the ointments of each solvent extract. 
Results: The antimicrobial activity of optimized formulation was showed significant activity against the tested bacterial pathogens. 
Conclusion: The present study confirmed the antimicrobial activity of the formulations. Furthermore, it can use as a potential dosage form for clinical utility. The results are the justification for the use of the plant in folk medicine. 
 
 
  
 



INTRODUCTION
The bacterial infections mainly occur with the injuries or ulcers or after the surgical treatments. Antibacterial substances help to inhibit or kill bacterial cells [1]. Infectious diseases are an increasingly greater extent in recent years. Furthermore, antibiotic-resistant becomes a major therapeutic problem [2]. The infectious diseases killing 50,000 people every day, and now, this is a world-leading cause for premature death [3]. Natural products of higher plants may possess a new source of an antimicrobial agent with a possible novel mechanism of action [4,5]. The researchers reported the antimicrobial activity of many plants and secondary metabolites of plants such as alkaloids, tannins, terpenoids, and flavonoids mainly responsible for these activities [6].
Medicinal plants used for the antimicrobial treatment started in ancient times. Several medicinal plant extracts and its phytochemicals have shown activity against all types of microorganisms including both Gram-negative and Gram-positive bacteria [7].
Ointments are semisolid preparation. They may or may not be containing medication used for the external application. Medicated ointments are intended to be applied externally in the body or to the mucous membrane. Non-medicated ointments commonly used as a base for the preparation of medicated ointments or used as such for lubricating or emollient effects. Plant drugs can also be formulated in the form of ointment [8,9]. The effective ratio of active ingredients incorporating with ointment base by trituration and after completion of the formulation, the ointment quality is assessing in terms of diffusion, irritancy, stability, and spreadability [10].
Eupatorium glandulosum popularly known as cat weed, Nilgiri weed, goat weed, or Mexican devil belongs to the family Asteraceae. E. glandulosum grows up to 1-3 m in height and it as an erect herb Silpa et al. and pestle. The prepared ointments filled into the tube and stored at room temperature [14][15][16]. The formulation from different solvent extracts of E. glandulosum is tabulated in Tables 1-3.

Evaluation of ointments
Organoleptic parameters such as color, odor, and texture were inspected through visual inspection.

Determination of pH
Pre-calibrated digital pH used for the determination of pH of ointments. One gram of ointment dissolved in distilled water (100 ml) and keeps it aside for 2 h. Standardization of the pH meter done with possible buffer solutions. Measurement of each sample solution done triplicate. The average values calculated and noted [17][18][19].

Spreadability
The spreadability of formulations determined using apparatus consists of a wooden block having a pulley at one end with a fixed glass slide on the block. Around 3 g of ointment placed on the ground plate and in between these plates and another glass plate having the same dimension of the fixed ground plate, the ointment sandwiched. On the top of these two plates, 1 kg weight placed for 5 min to expel air and to provide uniform of the ointment between the plates. The excess of ointments scrapped off from the edges. Two hundred and forty grams pull off with top plate and time required by the top plate to cover a distance of 10 cm noted. The spread capacity expressed in terms of times in seconds taken by two slides to slip off from ointment placed in between the slides under the direction of a certain load [17,[20][21][22]. The spreadability determined using the equation,

S=m×l÷t
Where, S -Spreadability m -Weight tied to the upper slide l -Length of glass slides t -Time is taken to separate the slide.
Separately measured the spreadability of each ointment and repeat 3 times for getting exact values and the average was taken as a consideration.

Viscosity
The viscosity of the formulation was done using Brookfield viscometer. The test sample is taken into a dry and clean 250 ml of the beaker. The viscosity of the sample determined at 25°C using viscometer. The viscosity measured in cps [18,23].

Homogeneity
Based on their appearance, all the developed ointments were tested for homogeneity by visual inspection [18].

Tube extrudability
It is a common test to obtain the force required to extrude the substance from the tube. The method applied for finding the applied shear in the region of rheogram corresponding to a shear rate exceeding the yield value and exhibiting consequent plug flow. In the present study, the method used for evaluating ointment formulation for extrudability based on the quantity in percentage ointment and extruded the ointment from the tube on a single application of pressure. More quantity of ointment extruded considered a better extrudability. The clean lacquered aluminum collapsible tube used to fill the ointment and the tube has a nozzle tip of 5 mm opening and applied the pressure on the tube with the help of a finger. It depends on the amount of ointment extruded from the tip when an external pressure applied on the tube is considered for the determination of the tube extrudability [17,18].

Antimicrobial studies
Bacillus subtilis and Escherichia coli used for the determination of the antimicrobial activity of various ointment formulations and standard cup plate methods used for the study. The nutrient agar solution poured into the previously sterilized Petri dishes up to 5 mm thickness. Each microorganism added to Petri dishes with a sterilized loop and plates were allowed to solidify for 5 min. On each Petri dish, five perforations made with a metal tube with a 4 mm diameter to receive test materials. The standard (neomycin sulfate ointment) and test materials (prepared formulations) added immediately into the wells and kept for incubation at 37°C for 24 h to allow the microorganism to grow and reagents to diffuse through the culture medium. At the end of the incubation, the zone diameter measured with the help of a zone reader [17,24,25]. All the experiments repeated 3 times to get accurate results.

Plant material extraction
The yield of alcohol, ethyl acetate, and chloroform extract of aerial parts of E. glandulosum was found to be 19.32% w/w, 10.85% w/w, and 7.24% w/w, respectively, as shown in Table 4.

Formulation of E. glandulosum ointments
The ointment from E. glandulosum prepared using the fusion method. The prepared ointment filled into the tube and stored at room temperature.   Silpa et al.

Evaluation of E. glandulosum ointments
The evaluation of ointments such as organoleptic parameters, homogeneity, spreadability, viscosity, pH, and tube extrudability results is shown in Table 5.

Stability studies
The optimized formulations stability studies carried out at a different temperature and there is no marked change in the appearance of prepared ointments. The pH remains the same as the original pH of the ointments. The product is stable in the base of the ointment. The results are shown in Table 7.
Evaluations of the prepared ointments showed good results in spreadability, homogeneity, viscosity, tube extrudability, and antimicrobial activity and stable at different temperatures (4°C, 25°C, and 37°C). Based on these evaluation parameters and antimicrobial activity, it was found that the optimized formulations of FEA4, FA4, and FC5 were showing good results. When compared to these three formulations, FA4 showing more antimicrobial activity than the other two.

CONCLUSION
The study is concluded as the optimized formulations of different solvent extracts of E. glandulosum are efficient antimicrobial formulations and might be useful for related disorders. Further, isolation is required for the researchers to establish potential activities.

AUTHORS' CONTRIBUTIONS
We declare that this work was done by the authors named in this article Miss Silpa performed the experiment and collected the data; Hamsalakshmi helps in writing and designing the manuscript. Dr. J Suresh proofread the whole manuscript and suggested the necessary changes.