Development and Antifungal Assessment of a Tridax procumbens Linn Herbal Cream: An In Vitro Study

Fungal infections represent a considerable risk to human health, especially for individuals with compromised immune systems. The increasing prevalence of antifungal resistance, coupled with a scarcity of effective treatment options, highlights the urgent need for innovative and powerful antifungal agents ^[1,2,3,4]. Fungal pathogens possess the ability to manipulate the host immune response, either by suppressing or activating it, to enhance their survival. Infections caused by fungi such as Candida and Aspergillus can lead to life-threatening conditions in immunodeficient patients. Additionally, fungal spores may provoke allergic reactions, asthma, and various respiratory problems. Skin infections caused by fungi like ringworm and athlete’s foot can result in significant pain and discomfort. Agricultural diseases are primarily attributed to pathogens such as powdery mildew, rust, and leaf spot. Furthermore, Aspergillus and Fusarium species can produce mycotoxins that contaminate food supplies ^[2]. Severe pneumonia is often linked to Cryptococcus. Antifungal medications, including ketoconazole, miconazole, and econazole, are among the active treatments available. Tridax procumbens L is recognized for its chemical constituents, including flavonoids, alkaloids, tannins, carotenoids, and fumaric acid. These compounds exhibit beneficial properties such as anti-inflammatory, antimicrobial, antioxidant, anticancer, and anti-diabetic effects. This plant is particularly noted for its potent antibacterial activity, attributed to the phenolic compounds it contains ^[3,4,5,6].

MATERIALS AND METHODS

Materials required:

Tridax procumbens Linn was collected from medicinal garden of Gokaraju Rangaraju college of pharmacy, Bachupally, Hyderabad. Ethanolic extract of Tridax procumbens, absolute ethanol, bees wax, lanette wax, Cetyl alcohol, stearyl alcohol, stearic acid, white soft paraffin, methyl paraben, propylparaben, triethanolamine, propylene glycol, water ^[4,5,7,8].

Instrument required: Electronic weighing balance, Soxhlet extractor, Brookfield viscometer, laminar air flow, BOD incubator, autoclave, pH meter, hot air oven, magnetic stirrer, water bath ^[5,6,9,].

Apparatus required: Round bottom flask, condenser petri dishes, beakers, glass rod, and spatula.

Method of Extraction:

Following the collection and identification, fresh leaves of Tridax procumbens Linn (100g) were washed and shade dried for 24-48 hours. The dried leaves were crushed to coarse powder and placed in 1L Soxhlet thimble attached with round bottom flask. The coarse powder with 500ml of ethanol were taken in round bottom flask. The leaf extract was obtained by Soxhlet extraction for about 6hours. The extract was collected and solvent was evaporated on water bath at 50°C. Then further dried in desiccator ^[9,10,12,14].

Evaluation of Herbal Extract:

Phytochemical Screening:

Standard phytochemical test was carried out for qualitative analysis of phytochemicals including, flavonoids, alkaloids, tannins, were tested by standard test and confirmed ^[5,6,7,8].

Test for Alkaloids: Extract (0.5 g) was shaken in a steam bath the test involves adding Dragendorff's reagent (a solution of potassium bismuth iodide) to the extract. A positive result, indicating the presence of alkaloids, is the formation of an orange or orange-red precipitate ^[10,12,15]. 

Test for Flavonoids: Add a 10% lead acetate solution to an herbal extract. If flavonoids are present, a yellow precipitate will form. This test is a simple qualitative method for detecting the presence of these phytochemicals ^[9,10,14].

Test for Tannins: The Ferric chloride test is a method to detect tannins in herbal extracts. A positive result is indicated by a dark blue or green-blue color when a ferric chloride solution is added to the extract. The intensity of the color can also be used to quantify the presence of tannin ^[9,12,16].

An extractive soluble in alcohol:

100 milli-litters of 90% alcohol were added to a Stoppard conical flask containing five grams of precisely measured powdered medication. After being continuously in an electrical shaker, the mixture was allowed to macerate overnight. The weight and percentage of the extractive were then calculated after the filter was cautiously evaporated until it was dry ^{[10,14,16,17]}.

Alcohol Soluble Extractive: Extractive Weight/ Drug Weight X 100

Total Ash

A China dish was used to weigh three grams of the drug, burn it at a temperature of no more than 450 degrees Celsius until the carbon was gone, let it cool, and then weigh it again until it stayed the same for three readings. To calculate the percentage of ash the air-dried drug was used ^[10,12,18].

Total Ash = Wt. of ash /Wt. of drug x 100

Acid Insoluble Ash

The ash was obtained after boiling it for five minutes with 25 milli-litters of diluted hydrochloric acid in it. After that, the insoluble substance was collected in a Gooch Crucible, cleaned with hot water, and burned until its weight remained constant. The amount of acid-insoluble ash was calculated with respect to the drug that had been allowed to air dry ^[10,12,18].

Method of Preparation of Cream:

Cream was prepared by oil in water emulsion method. Required ingredients for oil phase, such as Stearic acid, stearyl alcohol, cetyl alcohol, bees wax, lanette wax, were weighed accurately in a beaker and heated at 70°C on a water bath by continuous stirring ^[6,8,12]. Now in another beaker propylene glycol, triethanolamine, wateris heated at 70°C on a water bath to prepare water phase and. Now oil phase is added to the water phase with continuous stirring. Optimum quantity of herbal extract added to this by continuous mixing by mechanical stirrer Methylparaben, propyl paraben, perfume was added after the formulation is cooled. And the formulation table is as follows:

Table1: Formulation table of Tridax procumbens Linn cream.

Evaluation of Topical Formulations:

• Organoleptic evaluation:

Visual inspection is performed for their physical appearance, colour, texture, phase separation, homogeneity, texture and homogeneity were tested by placing cream in between thumb and index finger and pressing it ^[18,19,20].

• Procedure of Phytochemical Test:
• Determination of viscosity:

Viscosity of different formulations was determined using a Brookfield viscometer using spindle S-04 at 20 rpm ^[18,19].

• Stability test:

Stability of the formulations were tested by placing the formulations in environmental stability chamber at 25-27°C for 14 days and creaming or coalescence is inspected. ^[5,16].

• Determination of pH:

Dissolve 1gram from different formulations in 10ml of distilled water and homogenised using magnetic stirrer pH was determined using digital pH metre.ph of formulation was noted and the average results were shown ^[17,18,19].

• Spreadability:

The spread ability was determined by applying 1g of each formulation on a glass plate over which another plate was placed. The sample was pressed between two plates. A weight of 100g was allowed to rest on plate for 5mins. The increased in diameter due to spreading of cream was noted ^[17,18,19].

Anti-Fungal Assay:

Test organism: Standard fungal strain of Aspergillus niger (grown culture).

Antifungal Assay of Tridax Procumbens Linn Extract:

The antifungal assay of ethanolic extract of Tridax procumbens Linn was evaluated using modified agar well diffusion assay. Sabouraud’s dextrose agar (30ml) was poured in sterile petri dishes(n=3) and allowed to solidify. Two wells (10mm diameter) were created in 2 petri dishes containing media with a cork borer, and plates were inoculated with standardized inoculum of Aspergillus niger using a cotton swab. The ethanolic extract of Tridax procumbens Linn(1mg) were added to the wells made in two petri dishes and another petri dish was kept as control. These petri dishes were labelled and were incubated at 25°C for 7days after 1hour room stabilization period. The diameter of the zone of inhibition was subsequently measured to assess antifungal activity ^[15,16].

Anti-Fungal Assay of Cream:

The antifungal efficacy of topical agents was evaluated using modified agar well diffusion assay. Sabouraud’s dextrose agar (30ml) was poured in sterile petri dishes(n=4) and allowed to solidify. Three wells (10mm diameter) were created in 3 petri dishes containing media with a cork borer, and plates were inoculated with standardized inoculum of Aspergillus niger using a cotton swab ^[12,13]. The topical formulations (1mg) were added to the wells made in three petri dishes and another petri dish was kept as control. These petri dishes were labelled and were incubated at 25°C for 7days after 1hour room stabilization period. The diameter of the zone of inhibition was subsequently measured to assess antifungal activity ^[12,13,18].

RESULTS AND DISCUSSIONS:

Phytochemical analysis: Extract of tridax was used to perform standard phytochemical test and the results are given below in table 2.

Table 2: Results of phytochemical analysis.

Figure 1: Standard chemical tests for tridax procumbens.

Extractive solubility analysis:

Table 3: Results for Extractive solubility analysis.

Evaluation of Physical Parameters of Prepared Formulations

The physical characteristics of three formulations are presented in the table below. The results of creaming, coalescence test indicated that the formulations exhibit excellent stability and aesthetic appearance and uniformity.

Table 4: Evaluation of physical parameters of prepared formulations.

In Vitro Anti-Fungal Activity Study of Tridax Procumbens Linn Extract

The antifungal assay of 100ppm and 200ppm extract was performed and the results of zone of inhibition are as follows:

Table 5: Antifungal activity of herbal extracts (Zone of inhibition)

Figure 2: Zone of inhibition of extract against Aspergillus niger

Anti-Fungal Activity Study:

The antifungal assay of 3 formulations was performed and the results of zone of inhibition are as follows:

Table 6: Antifungal activity of herbal creams (Zone of inhibition)

Figure 3: Zone of inhibition of different formulations.