Preparation and Evaluation of Ant Repellent Herbal Gel Containing the Extract of Tagetes Erecta

On a global scale, herbal medicines are a significant part of health care systems. For medical care, almost 75% of the worldwide population depends on or trusts herbs and herbal remedies due to their diverse classes of phytochemicals and range of pharmacological effects. Non-steroidal ant repellents, or synthetic opioids, are commonly used to treat a variety of inflammations, but they have serious adverse effects [1]. According to the survey, there are a variety of plants which deals with repellent properties dispersed throughout the plant kingdom. They can serve as attractants, deterrents, phagostimulants, antifeedants, or alter oviposition. They can also alter key metabolic processes and induce fast mortality. They may also disrupt the insect's life cycle in various ways or prevent its development. Furthermore, it has been shown that compounds from a variety of floral species can serve as antifeedants, toxicants, and repellents to a variety of Coleoptera (Insects) that prey on stored goods [2]. A few well-known examples are Ageratina adenophora, Azadirachta indica, and Tagetes erecta [3-5]. A gel is a system comprising at least two parts that is solid or semisolid and is made up of an expanded mass that is surrounded as well as permeated by a liquid. Gels and jellies are made up of few numbers of solids scattered among a vast volume of liquid. [6-8].  Long-lasting insecticide-impregnated nets, or LLINs, provide defense against insects that might irritate, itch, or cause inflammation. Some protection is offered by repellents like diethyltoluamide. Better personal protection against the dominant group of social insects belonging to the order Hymenoptera of class Insecta is thus required. Permethrin-impregnated clothing may be sprayed or dipped to defend against insects in laboratory settings. Military and recreational wearers have also utilized this kind of clothing to protect themselves against arthropod bites [1].

Fig.1 - Ants: A Model for Studying Insect Repellent Effects

These studies showed that ant-repellent clothing protected activity for outdoor workers against self-reported tick bites. This study conducted an open-labeled study performed by extracting natural herbal extracts, which are easy to access such as Ageratina adenophora, Azadirachta indica, and Tagetes erecta. Subject volunteers are found a protective efficacy over a short period of time. This study suggests that treated clothing had not lost its efficacy over a period. Ants often inhabit areas with dust or food and can even be found on decaying organic matter. Ant infestations are a common problem for homeowners. Despite efforts to clean a room thoroughly, ants can appear in unexpected places and initiate new invasions within the home. While poisons and chemicals are effective in controlling ants, they can pose significant health risks to children and humans [7]. A gel is easily applicable as well as easily removable from the cloth or any surface area by simple washing with water. The bases and compatible excipients maintain dermatological and nontoxic factors [8].  

MATERIALS AND METHODS:

Preformulation study:

To ensure the development product of a reliable, safe, stable, and efficient dosage form, preformulation studies are required. During this phase of development, we studied the physicochemical characteristics and incompatibility of the pharmaceutical ingredients and how they interact with different formulation elements [9,10].

Pharmacognostic investigation:

1. Collection and Authentication: For the collection of Tagetes erecta (Marigold), Ageratina Adenophora (Crofton weed), and Azadirachta indica (Neem), the fresh flower and leaves were collected at the flowering stage in September 2023 from the local community market, West Bengal state, India [11].
2. Organoleptic Characterization: Colour, odour, taste, and size of the leaf and flower were observed [8].  
3. Physicochemical Characterization: After botanical evaluation, the shade-dried plant material was subjected to kept for 3 weeks before going for the extraction process. size reduction was done to get coarse particles, and then passed through a sieve no. 40 to get uniform particles. Then, the uniform size was subjected to standardization with different parameters as per the literature [12,13].
4. Solvent selection: Choose a solvent system that is composed of 90% distilled water and 10% methanol or chloroform because this prevents oxidation and hydrolysis and contains more amounts of antioxidant moieties than other solvent systems. The solvent used for the extraction is menstruum [14].

Table 1. Organoleptic Characterization for Leaves and Flowers:

Extraction and Storage:

1. Pouring: After performing the initial steps and allowing the system to settle for a while, these two were placed on a Rotary Shaker which maintained 100 rpm and the required strength should be left in a closed flask for twenty-four hours, shaking regularly for the first six hours and then remaining upright for eighteen [15].
2. Filtration: The extract solution was filtered through Whatman filter paper and a Buchner Funnel, and the filtrate was collected again in a 250 mL conical flask. For rapid filtration, vacuum filtration is used [16].
3. Solvent Evaporation: The filtered solvent collects in a petri dish. These were then placed on a Hot plate for solvent evaporation. Temperature is maintained at approximately 80°C. These were gently heated until complete removal of the solvent. To remove the solvent completely, utilize 2 days, about 4 hours of each day [17].
4. Extract Collection: The dried and solidified extract was scraped out from the Petri Plates (used during the solvent evaporation process) using a sharp Scrapper [14].
5. Extractive Yields: The weight of the dried and scraped materials was measured to calculate the extractive yields, and these were then stored in a freezer for future usage. The extractive yields measured were 0.242g and 0.284g, 0.461 g of Azadirachta indica, Tagetes erecta, and Ageratina adenophora, respectively per gram of dry mass [18].

Moisture content:

The moisture content of taken crude drug for the herbal gel preparation is measured by the loss of drying method to prevent oxidation. To measure the moisture content also requires a weighing balance and a Hot air oven [11]

Method / Process: Take an empty porcelain dish or Petri dish to take the sample. Weigh the empty porcelain dish or Petri dish by using the weighing balance and note the weight on gram scale. Tare the weighing balance to get 0 value, then add a specific amount of crude drug sample. Note the initial desired weight on the gram scale. Placed the loaded drug sample in the drying chamber for a certain period of time. Check the weight after 15- or 30-minute intervals. After complete drying, the dry content gives a constant weight. Note the final weight on gram scale.

Moisture content =Intial weight-final weightInitial weight×100 %

Phytochemical Screening:

Phytochemical screening is essential in the study of medicinal plants because it helps to identify and understand the bioactive compounds present in plants.

1. Test for Tannins:

3 ml of water extract of the sample in separate test tubes. After that, in the sample solution, 2 mL of 5% ferric chloride (FeCl₃) solution was added. Any change in colour or precipitate formation was observed [19].

2. Test for Saponin:

1-2 ml aqueous extract sample in separate test tubes. 5 mL of distilled water was added to each sample. Test tubes are shaken vigorously for 5 minutes and allowed to stand for 20 minutes. After that observed whether the honeycomb froth (foam) layer is present or not, which indicates the presence of saponin [11,20]. 

3. Test for Carbohydrates:

2 ml aqueous extract sample in separate test tubes. 2 mL of Benedict reagent was added to each sample. Then that solution was heated in a boiling water bath for 3-5 minutes. Any change in colour or precipitate formation was observed [11,21].

4. Test for Alkaloids:

Mayer’s reagent test: Take 2 mL of the aqueous extract sample in test tubes. A few drops of Mayer’s reagent (potassium mercuric iodide) were added to each sample. Any change in colour or precipitate (White / pale yellow) formation was observed [11,12].

Wagner’s reagent test: Take 2 mL of aqueous extract sample in test tubes. A few drops of Wagner’s reagent (2g of iodine and 6g of potassium iodide in 100 ml of distilled water) were added to each sample. Any change in colour (reddish) formation was observed [11,12].

5. Test for Phenolics:

To the 3 mL aqueous extracts, a freshly prepared aqueous solution of Ferric Chloride (FeCl₃) was added and observed for any colour change. [11,22]

6. Test for Flavonoid:

2 ml of the aqueous extracted sample solutions were taken in separate test tubes. Add prepared sodium hydroxide (NaOH) solution. If a yellow colour is observed, then add concentrated hydrochloric acid (HCl). Colourless sample solution indicates the presence of flavonoid groups [11,23].

7. Test for Glycoside:

Take 1 ml of aqueous sample in a test tube. Added 5-10 ml of dilute Hydrochloric acid (HCl). Then that solution was heated in a boiling water bath for 10 minutes.  After that, carbon tetrachloride and an equal amount of ammonia were added. Shake well to mix properly. A sample solution colour change to pink or red indicates a positive result [11,18].

8. Test for Coumarins:

2 ml of water extract of the sample in separate test tubes. After that, in the sample solution added 3 ml of 10% Sodium hydroxide (NaOH) solution was added. The sample solution changes colour to yellow indicating the presence of coumarins [11].

1. Test for Quinones:

Take 2 ml of water extract of the sample solution with 1 ml of concentrated sulphuric acid(H₂SO₄) solution. The sample solution's colour changes to red indicates the presence of quinones [23].

10. Test for Anthocyanins &Betacyanin:

Take 2 ml of water extract of the sample in each separate test tube. After that, in the sample solution 1 ml of 1N Sodium hydroxide (NaOH) solution. A sample solution colour change into blue or bluish-brown colour indicates the presence of anthocyanins and betacyanin’s [24].

Herbal Gel Preparation:

Two gelling agents were utilized in the formulation process at two different concentrations Azadirachta indica 20%, Tagetes erecta 50%, and Ageratina adenophora 30%, resulting in the manufacture of two distinct batches of gels. Both Xanthan gum and Carbopol 934 were used as gelling agents in this instance.  Preparation of gel with Carbopol 934: accurately measured four grams of Carbopol 934 and mixed it with 50 ml of purified water. After setting the beaker aside to allow the Carbopol to expand for 30 minutes, stir the mixture with a mechanical or lab stirrer set at 450 rpm for 20 minutes. Add extract aqueous solution, approx. 20 ml, then slowly pour 2 ml of glycerol mix and fill it with weighed liquid paraffin, stirring thoroughly. After all of the Carbopol had dispersed added the remaining distilled water was added, drop by drop triethanolamine was added to the formulations to balance pH (4.8–6) and get the gel at the desired consistency. Preparation of gel with Xanthan gum:  A beaker containing 3 g of Xanthan gum was filled with 50 ml of distilled water. Set aside the beaker to let the Xanthan gum swell for ten minutes and then stirring should be done continuously to prevent floc formation. Take 5 ml of propylene glycol and the small amount of extract into a separate beaker and thoroughly mix in the propyl and methylparaben. Then add glycerine as a preservative. Mix it properly and allow it to stand for 6 hours to get a semi-solid concentration.

Evaluation and standardization of gel:

1. PH: Using a digital pH meter, the formulation's conductivity and pH were measured in millivolts (mV). The glass electrode was calibrated using the equipment-specific solutions (pH of 4.00 and 7.00). The preparation was allowed to reach equilibrium while being measured for approximately five minutes. Average values were computed when the formula's conductivity and pH were analysed in triplicate [9].
2. Appearance and Homogeneity: A visual examination was used to assess the generated individual and polyherbal gels for homogeneity and physical appearance (colour, odour, etc) [25].
3. Viscosity: Using a Brookfield viscometer, the prepared herbal gels' viscosity was determined. Spindle No. 6 is used to rotate the gel at 100 revolutions per minute. ^[17]
4. Spreadability: Spreading 0.5 g of the gel over a circle between two horizontal planes with a 2 cm diameter pre-marked on a glass plate allowed for the measurement of the gel's spreadability. A second glass plate was then used. Five minutes were allowed for a half-kilogram of weight to lie on the upper glass plate. After the gel was spread, the circle's diameter was measured. [26]
5. Extrudability: Standard capped collapsible aluminium tubes were filled with gel compositions, and the ends were sealed with a crimp. The tube weights were noted. The tubes were clamped after being positioned between two glass slides. After covering the slides with 500 grams, the cap was taken off. Weighing was done on the amount of extruded gel that was collected. The extruded gel's percentage was determined as follows: >80% extrudability is good, >90% extrudability is excellent, and >70% extrudability is fair [25].
6. Stability study: The gel stability investigations are conducted at 37°C for 3 months. [27]

In vivo insect repellent study:

By 2013, the EU had made it a priority to stop using animals in cosmetics testing, providing human in vivo research a boost, especially non-invasive research. [28] Because of this, the study researchers paid close attention to a variety of imaging methods, including confocal microscopy, tomography, and fluorescence investigations [1, 3-5]. Ant is selected as a candidate to perform this study. Due to their jointed legs and exoskeleton, ants belong to the phylum Arthropoda. They have colonized almost every continent on Earth, making it simple to gather data using the manual technique. Ants transport food particles and frequently return them to their nest or mound. Sugar powder is used to gather and sample. Ant bites and stings may result in swelling, irritation, allergic reactions, and in extreme cases, anaphylaxis. This collection is a little dangerous. A clean cotton cloth sample (10 cm × 10 cm) was taken and cut into 3 pieces. Each and every piece was equal in size. Those clothes were kept in a chamber without contact with observers at room temperature. In the centre, sugar powder was kept to attract ants. The experimental chamber maintained environmental conditions equivalent to atmospheric pressure, temperature, humidity, etc. Herbal gel was applied to different sides and corners of the cloth, and after 5 minutes to dry, leave it. The different batch formulation is compared with the standard marketed repellent and blank (in which no repellent is applied). Ticks can either enter or remain on the surface treated with plant extract gel. Tick counts were recorded after 10 min.

RESULTS:

Table 2. Extractive values for Leaves and flowers:

Table 3. Moisture content:

Table 4. Phytochemical screening [11]:

Table 5. Physical Observations of Compatibility Study:

Table 6. Physiochemical Analysis of Herbal Gel:

Table.7: Data of % ant repellence values of herbal plant extracts at 15 min intervals

Table.8: Data of % ant repellence values of herbal plant extracts combination at 15 min intervals

Fig. 2 - Ant repellent study conducted by an in vivo study on a cloth using different formulations: a) marketed product, b) blank formulation, c) F₁ formulation, d) F₂ formulation