Formulation And Evaluation of Antimicrobial Cream Form Leaves Extraction of Mulberry and Tridax Procumbenss.

The World Health Organization states that the best source of a variety of drugs would be medicinal plants. Traditional medicine, which contains substances derived from medicinal plants, is used by almost 80% of people in developed countries. Since they both have antibacterial properties, plant extracts and phytochemicals can be extremely beneficial in therapeutic therapy. The antibacterial properties of many plants, which are connected to compounds produced in the plant's secondary metabolism, have led to their use. These items are identified by their active ingredient, such as the phenolic chemicals found in essential oils¹.

The demand for cosmetics is primarily influenced by the availability of herbal cosmetics. The public has been giving increasing attention to herbal formulations due to their superior qualities and lower incidence of side effects. 1. It additionally supplies the skin with the necessary nutrients and hydration (Mali AS, et al., 2015). 2. In essence, the herbal cream is an oil and water emulsion. The natural ingredients chosen for preparation of herbal cream are mulberry and Tridax Procumbenss linn. The choice of these ingredients is based on their individual properties².

Mulberry

Mulberries, which belongs to the Moraceae family, grow in a variety of climates and environments, ranging from tropical to temperate. The Moraceae family of flowering plants, frequently referred to as the mulberry or fig family, comprises about twenty-four species, each of which contains at least a hundred variations and one subspecies. The name Morus is derived from the Latin word "mora," which means "delay," which is probably due to the sluggish growth of its buds. Mulberries or their extracts are used to treat a range of acute and chronic conditions because of their antibacterial, anti-hyperglycemic, anti-hyperlipidemic, anti-inflammatory, and anti-cancer qualities. Morus species' fruits, leaves, twigs, and bark all have substantial anti-tyrosinase inhibitory activity, making them a good choice for use as a whitening ingredient in cosmetics³.

• Mulberry (Morus) belongs to the Moraceae plant family and includes several species, such as the black mulberry (M. nigra), red mulberry (M. rubra), and white mulberry (M. alba).
• Native to China, this tree is now cultivated in many regions, including the United States, Europe, Asia, and Africa.
• Mulberry leaves have a variety of culinary, medicinal, and industrial applications.
• Mulberries are the fruits of mulberry trees (Morus sp.) and related to figs and breadfruit.
• The trees are traditionally grown for their leaves mainly in Asia and North as they're the only food that silkworms eat.
• They carry colourful berries most commonly black, white, or red that are often made into wine, fruit juice, tea, jam, or canned foods, but can also be dried and eaten as a snack⁴.

Figure 1. Mulberry

Tridax Procumbenss

Tridax Procumbenss Linn. (figure 2) commonly known as coat button is a perennial plant belonging to the family Asteraceae⁵. It is a native to the tropical Americas, but it has been introduced to tropical subtropical, and mild temperate regions worldwide, it is often rooting at node solitary ,long stalked, yellow composite, heterogamous, bisexual flower with white flowing heads and very hairy, with coarsely toothed, petiolate, ovate or lanceolate leave whole arial part is useful medicinally, leaves possess wound healing, insecticidal, antisecretory and hypotension action, while seeds are used to control bleeding Tridax Procumbenss is widely distributed in India up to 2400m. above sea level⁶. The leaves have medicinal value and used to treat catarrh, dysentery and Diarrhoea, the different leaf extracts are used to Antiseptic to treat fresh cut wound, burns in anaemia. It contains flavonoid, alkaloid, carotenoid, cinnamates, liganans, benzoic acid derivate, phytosteroid tannin, crude proteins fibbers, soluble carbohydrates and calcium oxide. Due to its widespread distribution and abundant seed production, it has significance as a weed⁷. The week-straggling herb Tridax Procumbenss is 12–24 cm long, with a few leaves that are 6–8 cm long and very long, slender, solitary peduncles that are at least a foot long. Tridax has two flower types: disk-florets and ray-florets. Cypsela is the basal placentation fruit. The leaf juice is put directly to the wound. In traditional medicine, its leaf extracts are used to treat infections and skin-related issues. It has pharmacological activity as antimicrobial activity ⁸_.

Figure 2. Tridax Procumbens

Antimicrobial Activity:

Heat stable proteins of morus alba tested for the antibacterial activity against Escherichia Coli, and compared with the antibiotics⁹. In another study chloroform and ethanol sequential leaf extracts of morus alba at different concentration of the extract were tested for antimicrobial activity against various bacterial strains. The zone of inhibition was determined against the microorganisms.

The effect of these extrscts were compared to standard drugs, results of the antimicrobial activity revealed that the extracts showed noticeable antimicrobial activity in dose dependant manner against the organisms studied^10,11.

MATERIALS AND METHODS:

Soxhlet Extraction:

Soxhlet extractor was invented in 1879 by Franz von Soxhlet, a German chemist. The original use of this apparatus was the extraction of a lipid from a solid material. It is a component of equipment used in scientific settings to recover compounds that have limited solubility in a solvent and impurities that are insoluble in that solvent¹².
Using a solvent and an extractor, this traditional solid/liquid extraction process involves extracting directly from the raw material. In this kind of extraction, the solid sample is ground up and put within a cotton-topped, porous cartridge. This cartridge is located in the chamber of the Soxhlet extractor. At the same time, the solvent to be used is introduced into a flask and heated, so that the condensing vapours fall on the cartridge containing the sample, extracting the soluble compounds. This technique is easy to perform and does not require any additional filtration step. However, it requires a long extraction time and large amounts of expensive and dangerous solvents, as well as not allowing stirring¹³.

The operation cycle is the following:

The dry sample is placed inside the cartridge and then paced into the Soxhlet extractor with a siphon. Meanwhile, the solvent located inside the distillation flask is heated until it reaches its boiling point thanks to the heat source. At this stage, the vapor ascends through the flask's neck, passes through the vapour tube, and arrives at the condenser. At this point, the vapor condenses and returns in liquid form to the extractor. In the cartridge area, the solvent droplets are gathered and come into contact with the sample. The solvent and the chemicals of interest are in existence at this time. Simultaneously with the extract, the extractor collects the solvent until the extract is concentrated enough to flow into the flask through the siphon. The solvent is recycled several times extracting in each cycle a fraction of extract. The desired compound is more concentrated in each cycle and the colour of the solvent in the flask is darker as well due to this reason. After the extraction, the solvent is removed by evaporation getting only the desired compounds¹⁴.

Collection of plant material:

Tridax Procumbenss Linn and mulberry plants were collected in the month of April 2024 from the home garden. The leaves were washed and air dried. Authentication was done by P. J. wagh sir (Anand Niketan College of Science, arts and commerce, Warora)

The leaves of Tridax Procumbenss and mulberry were dried at room temperature and dried leaves were crushed to coarse powder through mortar and pestle¹⁵.

Chemical:

The chemical solvents and other products that have been used in the experiments and methods are the following:

• Distilled water
• Syringe filter
• Ethanol (C2H6O) was supplied by Molar Chemicals Kft, purity: 99.5%.
• Chloroform (CHCL3) was supplied by Molar Chemicals Kft, purity: 99%.
• Diethyl Ether (C2H5) was supplied by molar chemical Kft, purity: 98%.¹⁶

Method of extraction:

Approximately 30 g was measured for this operation and put into the paper thimble inside the chamber. Additionally, 240 millilitres of solvent were added to the solvent flask. Three polarity-varying solvents were used: 96% ethanol, ether, and chloroform. The round-bottom flask containing the solvent was submerged in silicon oil, and the Soxhlet apparatus was attached to a cooler. In order to heat the solvent and begin the extraction process, the condenser's cooling water was turned on first, followed by the silicon bath's heating element. The extraction process ended when the solvent in the Soxhlet apparatus turned transparent once more.

It took approximately three days (18 hours) to extract the raw material with diethyl ether, one day with chloroform, and four days (24 hours) to remove the raw material with 96% ethanol.
To obtain the extract without solvent, the liquid must evaporate after the Soxhlet extraction is complete. This involves implementing a control valve and a vacuum pump to evaporate each flask in the rotary evaporator. Only the extract that was produced as a product after this stage is still in the flask and is gathered in a sample bottle. After being dried, the removed material was thrown away. The condensed and evaporated solvent is gathered in a different flask and put back into a bottle for later use.

The total weight of the extract and the dry raw material must be determined in order to calculate the yield. The weighted mass of the seed and the dry content of the raw material were used to determine the mass of dry raw material. The mass of the flask containing the extract and the mass of the flask empty were used to calculate the mass of the extract¹⁷.

Figure  4. Extraction Of Mulberry

? Infusion: Tridax Procumbens

Water is poured over the medications, and after a particular period of time—typically 15 minutes—it is allowed to remain in contact with the water, stirring occasionally and the liquid is filtered off¹⁸.

Figure 5. Tridax Infusion Extract

PREPARATION OF CREAM –

PROCEDURE:

Every ingredient was precisely weighed. After melting beeswax in a porcelain plate, liquid paraffin was poured. Olive oil was added to the above-melted base after homogenisation. Plant extract was used to dissolve borax in a suitable amount of water, which was then heated. Drop by drop, the water was vigorously stirred into the greasy area. To get the proper consistency, the molten mass was then allowed to cool¹⁹.

Figure 6. Formulated Cream

FORMULATION COMPONENT:

Table 1. Formulation Table of Herbal Anti-Microbial Cream

EVALUATION OF CREAM -

1. Evaluation of cream pH of the cream

Calibration of the pH meter was done using standard buffer solutions. The pH of the cream was determined by dissolving and balancing around 0.5 g of it in 50.0 ml of pure water. A precise measurement of 5 ± 0.01g of the cream was made in a 100ml beaker. The cream was spread over 45 millilitres of water. The pH of the suspension was estimated to be 27°C using the pH meter. The pH of the cream was determined to be between 6 and 7.5, which is good for the skin's pH. The pH of each formulation was closer to that of the suitable skin ²⁰.

2. Organoleptic evaluation

The resulting cream's organoleptic qualities, including colour, odour, and condition, were evaluated. The cream's colour and roughness were used to measure and grade its appearance²¹.

3. Dye test

The efforts of Dhase and his team in 2014 was the source of this procedure. It blends the cream with the fiery red colour. Examine the cream under a microscope after placing a drop of it on a covered microscopic slide using a cover slip. If the dispersed globules appear crimson, the ground is colourless. It's a sort of o/w cream. The converse situation arises in the w/o form cream, when the dispersed globules appear colourless against the red ground²².

4. Homogeneity

The uniformity of the formulations was assessed both visually and tactilely²³.

5. Spreadability studies

A specialised tool has been created to assess the formulations' spreadability. The duration in seconds it takes for two slides placed far apart from the formulation to slide under the application of a specific load is known as spreadability. Two standard-sized glass slides were chosen. One of the slides was covered with the formulation whose spreadability needed to be ascertained, and the other slide was positioned on top of the formulations, which were jammed together along the slide's 5 cm length. To create a thin layer, the formulation between the two slides was uniformly compressed by placing a 100 g weight on the upper slide. After the load was lifted, the silence. After the load had been removed, the formulation that was stuck to the slides was scraped off, and the slide that had the formulation on it was fixed. Over it was the second movable slide, one end of which was fastened to a string that could be loaded using a pan and a basic pulley. The time it took for the upper slide to move 5.0 cm and separate from the lower slide under the load's direction was recorded after a 30g weight was put on the pan²⁴.

6. Antimicrobial test:

Protocol: The media utilised was nutrient agar. A culture of Staphylococcus aureus microorganisms was employed. A 24-hour incubation period was built up.

Method: Diffusion of agar bore well.

Procedure: 40ml of sterile nutrient agar media was added to each sterilised plate after Escherichia coli (Gram +ve bacterium) suspension was added. To ensure that the agar and the test organism were mixed uniformly, the plates were gently shaken. The plates were allowed to solidify on a level surface. A cork borer was used to bore a 1 cup, 10 mm diameter hole in the middle of each plate. A sterile dissecting needle was used to remove the agar discs, taking care not to harm the cups.Equal amounts of cream formulations of the same strength were put in each cup, and the plates were incubated for 24 hours at 37°C +/- 2°C. The entire procedure was performed in an aseptic environment, and the zone of inhibition was determined. Figure 8.25 displayed the zone of inhibition for the produced formulation²⁵.

RESULT:

QUALITATIVE TESTS FOR PHYTOCHEMICAL SCREENING:

1. Table 2. Detection of alkaloids

2. Table 3. Detection of carbohydrates

3. Table 4. Detection of reducing sugar

4. Table 5. Cardiac glycoside

5. Table 6. Detection of flavonoids¹⁶

6. Table 7. Detection of phenolic compound

7. Table 8. Detection of tannins