DEVELOPMENT AND EVALUATION OF LAWSONE (HENNA) NANOEMULSION FOR ANTIFUNGAL AND ANTI-BACTERIAL ACTIVITY

DEVELOPMENT AND EVALUATION OF LAWSONE (HENNA) NANOEMULSION FOR ANTIFUNGAL AND ANTI-BACTERIAL ACTIVITY

Nidhi Gupta, Krishna Kumar *, Neeraj Kumar and Amresh Gupta

Samarpan College of Pharmacy, Lucknow, Uttar Pradesh, India.

ABSTRACT: Nanoemulsions, also mentioned as submicron emulsions, ultrafine emulsions and miniemulsions, are submicron-sized colloidal particulate systems considered as thermodynamically and kinetically stable isotropic dispersions; they contain two liquids like water and oil (immiscible), stabilized by an interfacial film consisting of a suitable surfactant and co-surfactant to form a single phase. In this research, lawsone nanoemulsion was developed by using Neem oil, tween 80 and ethanol. The formulation optimization was done by designing a ternary phase diagram followed by water dilution method. We developed 6 formulations (F1-F6) with different concentrations of oil and surfactant but drug concentrations are the same in all developed formulations. Developed formulations are evaluated for different parameters like pH, solubility, drug content, viscosity, transparency, size of globules and stability study, etc., and found to be appropriate in all tests. Since, oil/water Nanoemulsion had greater density than water/oil systems, the stiffness values increased significantly whenever the water content increases or when the system switched to oil/water type. Antimicrobial screening of the drugs and formulations were also screened against bacterial and fungal spices and got best results. The data shows that the particle size reduces as the ratio of oil to surfactants: co-surfactants increases. As a consequence, Nanoemulsion particles settled close to the body.

Keywords: Nanoemulsion, Antimicrobial, Ternary phase diagram, viscosity

INTRODUCTION: Nanoemulsions, also mentioned as submicron emulsions, ultrafine emulsions, and miniemulsions, are submicron-sized colloidal particulate systems considered as thermodynamically and kinetically stable isotropic dispersions they contain two liquids like water and oil (immiscible), stabilized by an interfacial film consisting of a suitable surfactant and co-surfactant to form a single phase.

A number of surfactants with diverse characteristics (ionic or non-ionic) had been used with such Nanoemulsion. Most widely used among them were nonionic surfactants (sorbitan esters, polysorbates), anionic surfactants (potassium laurate, sodium lauryl sulphate), cationic surfactants (quaternary ammonium halide) and zwitterions surfactants (quaternary ammonium halide).

Early nanoemulsions were oil-in-water (O/W) type emulsions with average droplet diameter ranging from 50 to 1000 nm. Nanoemulsions more recently are classified into three categories such as O/W type (oil is dispersed in aqueous phase), water-in-oil (W/O) type (water is dispersed in oil phase), and bi-continuous (microdomains of water and oil are interdispersed within the system). Transformation among these three types can be attained by altering the components of the emulsions. Multiple emulsions are also nanoemulsions, where both O/W and W/O emulsions are present simultaneously in one system. For stabilizing these two emulsions, both hydrophilic and lipophilic surfactants are used simultaneously. Nanoemulsion offer various advantages over other dosage forms and these advantages are (1) increased rate of absorption, (2) reduced variability in absorption, (3) protection from oxidation and hydrolysis in O/W nanoemulsion, (4) delivery of lipophilic drugs after Solubilization, (5) aqueous dosage form for water-insoluble drugs, (6) enhanced bioavailability for many drugs, (7) ability to incorporate both lipophilic and hydrophilic drugs, (8) delivery systems to enhance efficacy while reducing total dose and side effects, (9) as non-toxic and nonirritant vehicles for skin and mucous membrane delivery and (10) release control by permeation of drug through liquid film, whose hydrophilicity or lipophilicity, as well as thickness, can be precisely controlled ¹.

Antibiotic-resistant bacteria are becoming more common, which may reduce conventional medications' effectiveness, making patient treatments more expensive and complex. The abuse & overuse of antibiotics as well as the usage of anti-bacterial chemicals in food-producing animals to enhance animal health & increase making, has resulted in the creation of resistant microbes ². In 2004, it was projected that more than 70% of hazardous bacteria were resistant to at most one commercially active antibiotic ³. Escherichia, Klebsiella and Pseudomonas are only a few instances of Gram-negative infections. Gram-negative bacteria are responsible for > 60% of hospital sepsis cases ⁴. Pseudomonas aeruginosa is responsible for over 80% of these bacterial infections. Furthermore, fungi are responsible for around 40% of deaths from nosocomial infections, with Candida and Aspergillus accounting for 80% of them ⁵. Antifungal drugs such as polyene antibiotics, fluoropyrimidine, Nystatin and the azole group of pharmaceuticals are presently available for the treatment of widespread mycosis. Antifungal treatment targets:  Fungicides work on a variety of targets. Polyene medicines, including Itraconazole, Posaconazole, & Clotrimazole, all work on the plasma membrane. The antifungal medication also targets DNA synthesis and includes medicines like Pyrimidine analogs, such as Flucytosine. Echinocandins Caspofungin acetate is an antifungal medicine that works on the cell wall.

Herbs have been employed in traditional herbal treatment in the past. Numerous plant-derived chemicals have been shown to exhibit anti-bacterial action and a large spectrum of active ingredients has been found. Numerous studies have discovered that bioactive chemicals from medicinal herbs have anti-tumor, anti-carcinogenic, anti-bacterial, anti-bacterial, antifungal, antioxidant, antiviral, anti-inflammatory, and anti-carcinogenic activities ⁶. Henna is utilized to cure a range of ailments in herbal medicine, including headaches, ulcers, rheumatoid arthritis, diarrhea, fever, diabetic ^{7, 8}. Furthermore, these herbs are employed to combat infection disorders. The plant's inhibitory activity against gram gram-negative, gram-positive, and fungal bacteria & fungi has been described. The existence of anthraquinones (lawsone) as key constituents of the plants accounting for roughly 1.5 percent of henna is credited with this effect ⁹.

Lawson is an organic dye used to color hands, fingers, nails, and hair in, Asia, Arabia, and different parts of Africa. It has a huge range of biological actions, including antimicrobial properties for hair & skin, antitumor, antimicrobial, anti-parasitic, cytotoxic properties, and anti-inflammatory properties. Many studies have shown that henna has antimicrobial properties, particularly against gram-positive bacteria, as well as antifungal action towards dermatophytes and wounds repair¹⁰. However, few studies have looked at its impact on nosocomial infections. The antagonistic capability of the Henna plant towards pathogenic bacteria that cause infectious diseases was also investigated.

Pathways for Delivery of Drugs through Human Skin: Drug particles in interaction with the surface of the skin can enter through three different routes:  hair follicles, sweat ducts & sebaceous glands (together known as the shunted or appendageal route), or immediately through the stratum corneum Fig. 1. Scientists have argued the relative relevance of the shunted or appendageal route against transportation over the stratum corneum for years and the lack of an appropriate experimental model to separate the three paths has made things even more confusing. The usage of hydrated skin or epidermis membranes in vivo investigations is common because the swell accompanied by hydration closes appendages. The pre-steady-State penetration of polar compounds and flux of large polar molecules or ions that have difficulties diffusing over the entire stratum corneum, according to scheuplein and colleagues, is caused by a follicular shunt pathway. The corneum of the stratum is composed of 10-20 levels of corneocytes from 10-15m while dry to 40m when wet. It consists of keratin-rich keratinocytes (bricks) imbedded in a fibrous tissue (mortar) mostly composed, phospholipids, lipids, sterol, triglycerides sulfate, and sterol/wax esters. Corneocytes are elongate, polygonal, and flat (0.2-1.5 m thick, 34-46 m in diameter) instead of brick-shaped, which is important to remember. Waters is an important constituent of the stratum corneum, acting as a plasticizer to prevent cracking and assisting in producing a natural moisturizing factor (NMF) that helps keep the skin supple. Hydrophilic substances are assumed to dissipate via the lipid matrix between intracellular keratin filaments, while lipophilic compounds are thought to dissipate through the soluble areas near the external surface of intracellular keratin filaments ¹¹ (intracellular or transcellular route) (intracellular route) Fig. 2

Structure of Nanoemulsion: Based on the quantities of the ingredients, a combination of oil, water, and surfactants can produce a wide range of morphologies and stages. In this aspect, the mobility of the surfactant film is critical. The presence of numerous diverse structures, such as droplet-like forms, aggregates, and bicontinuous constructions, will be enabled by a flexible surfactant layer, which will widen the range of Nanoemulsion presence. The presence of a bicontinuous structure will be hampered by an extremely stiff surfactant coating, which will limit the spectrum of existence. Depending on the component ratio, structural studies can indicate the presence of normal emulsifiers, anisotropic crystalline hexagon or cubic phases, lamellar structures, and Nanoemulsion. The interior architecture of a Nanoemulsion carrier is critical for phase conductivity and, as a result, drug diffusion in the corresponding phases.

The intricate phase behavior and varied microstructures seen in macroemulsion systems have piqued the interest of scientists ¹².

MATERIAL AND METHOD: The Lawsone (Drug) was obtained from Himedia Pvt. Ltd., Mumbai, India. Neem oil was used as an oil phase purchased from Devinez Pvt. Ltd. and fulfilled by Amazon. All other chemical used in this study was appropriate grades supplied by justified vendors. The equipment used in this study during work was electronic balance, UV/Vis spectrophotometer, Micropipette, autoclave, water bath, magnetic stirrer, incubator, homogenizer, Brookfield viscometer, digital pH meter, Vortex shaker, FTIR spectrophotometer, melting point apparatus, and thermometers, etc. The required apparatus such as beaker, volumetric flask, measuring cylinder, glass rod RD bottles, etc. were used for the experiment.

Preformulation Studies: Preformulation is a design phase in which researchers characterize the chemical, physical, and mechanical characteristics of the drug material to create a dosage form that is effective, durable, and secure. As a result, Preformulation experiments are required to describe the medicine so that the delivery of drugs can be properly designed. The following are some of the pre-formulation investigations that were carried out as part of this project:

• Melting point
• Solubility
• Uv-spectroscopy
• FTIR
• PH

Melting Point: Volumetric flask of one end closed is filled with enough dry powder to produce a 0.25cm long to 0.35cm column at the end of the hole that is tightly compacted as tightly as feasible by gentle hammering on a solid surface. The device is operated following industry best practices. The block is warmed until it reaches a temperature approximately 30° below the projected melting point. The capillary is put into the hot plate, and the temperature is raised at a rate of around 1 to 2°C per minute until the melting is finished. The start of melt is classified according to the temperature at which the sensor signal initially leaves its initial value, and the end of melt, or the melting point, is described as the temperature at which the sensor signal achieves its final value. Both of these temperatures are inside the melting range ¹³.

Solubility Studies: Solubility is the interaction of components of a mixture to establish homogeneous biochemical dispersion. At room temperature, 10 mg of sample was placed independently in 10 ml of several solvents in firmly capped tubes & shaken. The table shows the solubility characteristics of two medicines in several solvents Table 1 ¹⁴.

TABLE 1: SOLUBILITY PROFILE

pH: A pH meter with a glass electrode is used to determine pH. The power of H+ concentration in a solution is represented by pH. The equation that follows defines it. The natural log of the mutuality of H+ in dilute fluids well matches this amount.

The pH of Nanoemulsion compositions was determined utilizing an ELICO LI120 pH meter that had been adjusted before being used with a buffer solution at pH 4 & pH 9.2.

A specific quantity of the formulation was measured out, dissolved with measured filtered water, & thoroughly mixed. The electrodes of the pH meter were submerged in the pH formulation that had been created.

A specific amount of the mixture was measured, diluted with measured filtered water, and thoroughly mixed. For pH measurement, the electrodes of the pH meter were submerged in the produced formulation determination. A pH meter was used to determine the pH of around 2gm of formulation distributed in 20ml of purified water ¹⁵.

FTIR Analysis: Responsive units in a molecule are identified using FTIR. The drug was examined at 4mm/s with a precision of 2cm over a wave number range of 400 to 4000cm-1 on a KBr disc. The typical peaks were captured on film ¹⁶.

UV-spectroscopy: Lawsonia innermis was subjected to a UV spectroscopic test to determine its authenticity. Using the dilution procedure, the Lawsonia innermis was dissolved in ethanol in a 10 ml volumetric flask and diluted up to 10 times.

UV spectroscopy was used to establish the ethanol's maximum concentration of Lawsonia innermis. Lawsonia innermis standard curve in ethanol was also displayed.

Calibration Curve: The calibration curve was created by immersing one gram of Lawsonia innermis in 100 mL of ethanol as a stock solution. Using the serial dilution procedure, we create solutions with varying concentrations of Lawsonia innermis from a stock solution. As a result, the OD was measured at 426nm using a UV spectrophotometer ¹⁷.

Development of Nanoemulsion:

Optimization of Co-surfactants, Oils and Surfactants: Optimization of the concentration of surfactants, oils and co-surfactants was carried out using various ratios.

The ratio of surfactant and co-surfactant mixture (Smix) varies from 1:1, 1:2, 1:3 & 1:4. The ratio of the Smix and oil concentration is 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9.

Aqua distilled water is added by performing the titration and then stirred by using a magnetic stirrer till it converts to translucent and no phase separation ¹⁸.

Optimization of Formulation: Researchers have used numerous formulation & optimization strategies such as the ternary phase transition, Box-Behnken design, & D-optimization designs, according to diverse literature.

We used a ternary phase diagram Fig. 1 and a design of experiment technique to optimize the formulations in this investigation ¹⁹.

FIG. 1: TERNARY PHASE DIAGRAM

Nanoemulsion Formulation: Formulation of the Nanoemulsion was carried out by using the method known as the spontaneous emulsification method. Total 6 formulations were carried out, known as F1, F2, F3, F4, F5 and F6 Table 2. The Lawsonia was added to the oil phase consisting of standardized butylated hydroxytoluene; further, the Smix solution was added to it, which is a mixture of co-surfactant and surfactant, then allowed for stirred with the magnetic stirrer until a homogeneous mixture was prepared.

Then the water is added by using the mode of titration and then left for stirring condition until the Nanoemulsion will be formed which is noticeable by the development of a translucent solution ²⁰.

Protocol:

• Oil Phase: Neem oil was taken as an oil phase for developing Nanoemulsion.
• Aqueous Phase: Take distilled water and add tween 80 (surfactant).
• Lawsonia is dissolved in ethanol (co-surfactant) and mixed with an aqueous phase. Both the phases (aqueous and oil phase) were heated in a water bath up to 7°C.
• The aqueous phase was placed on a magnetic stirrer, and the oil phase was added dropwise into the aqueous phase to develop the coarse emulsion. Allowed the Coarse emulsion to stir on a magnetic stirrer for 20 min to get the Nanoemulsion.

TABLE 2: FORMULATION DEVELOPMENT

Evaluation of Developed Nanoemulsion: The following criteria were used to evaluate the Nanoemulsion after it had been formed:

Optical Transparency: Inspecting the specimen in a clean & visible vessel in the vicinity of good lighting versus reflecting into the eye and viewing against a dark and white lighted background was used to evaluate the formulation's optical transparency.

Viscosity: Using a Brookfield Viscometer, this process evaluates the fluid velocity. Viscosity is a measurement of fluid friction that can be thought of as the internal friction that occurs when one stratum of fluid moves about another. The ratio of combing force to the shear rate is measured by viscosity. Verify also that viscometer has been properly optimized If not, use software to calibrate. Similar to the Calibration Standards, the specimen container and quantity should be similar. Equilibrate the sample's temperatures with the temperature specified in the requirement. (±1°C). Utilizing the bubble level on the rear of the viscometer, double-check that it is level. The device with the spindles connected as well as the speed set as specified in the design specifications for the Brookfield LV-II. After ten min, the primary screen will flash 00.0.

Immersion of the appropriate spindle in the specimen to the notch on the spindles shaft as specified in the design specifications. Allowing air bubbles to form is not a good idea. Connect the viscometer to the spindle. The spindle must not contact the container's bottoms or edges and should be centered. Make sure the viscometer is appropriate. The spindle number 64 was revolved at 60 revolutions per minute. Nanoemulsion specimens were permitted to stay at room temperature for 30 minutes before readings were conducted ²¹.

Droplet size Test: Zetasizer was used to evaluate the best Lawsonia Nanoemulsion formulations. The specimen was inserted into this chamber and served as a delivery and control chamber. The chamber was then filled with a suitable solvent. A laser beam was now expected to slide on the sample cell. They were sent towards the sensor after completing the necessary amount of runs. The formula's particle diameter range and the mean particle size can be determined using this information. A particles analyzer can be used to estimate the mean particle size of Nanoemulsion formulations ²².

Phase Separation Test: The Nanoemulsion stability was assessed using the centrifugal test. About 4-8 g of the finished Nanoemulsion composition was placed in a test tube and agitated at 5000 rpm for around 10 minutes at 250°C in this test. As a result, the Nanoemulsion gel was visually examined for any signs of phase separating ²³.

Anti-bacterial and Antifungal Analysis:

Requirements: Micropipettes, microbiological growth, extract operating fluids, conical flask, tips, marker, Petri plates, nutrient agar medium, potato dextrose agar media, nutrient agar media, sterile nutrient agar media, nutritional agar media, and nutrient broth media.

Principle: The antifungal testing of medicinal plants against test pathogens was done using the agar well diffusion method.

Procedure:

• Nutrient agar medium was made and placed into sterile plates after being autoclaved (sterile).
• After that, the media was allowed to set.
• In each Petri plate labeled Escherichia coli and Staphylococcus aureus (Bacterial species), 20 l of the pathogen was disseminated (Fungal species).
• Using sterile tips, 4 wells of 8mm diameter were bored after 5-6 minutes of spreading.
• Then, using a micropipette, 50 l of the extract was put into each well.
• The plates were then incubated for 24 hours at 370°C ²⁴.

In-vitro Release Study: A customized Franz diffusion (FD) cell was used for the in vitro release of drug testing. Between the donor and receptor compartments of the FD cell, a dialysis layer was positioned, and approx. 2 g of the optimized Lawsone formulation was applied on the dialysis membrane. Phosphate buffer (PH 5.4) has been used as a dissolution medium. The cell's temperature was kept constant at 37°C using flowing water jackets and a mechanism. The intended apparatus was mounted on a magnet stirrer, and magnetic beads were used to continuously mix the dissolving media.

After that, the samples (5ml) were taken from the receptors chamber at appropriate intervals and replaced with an equivalent volume of a new medium. The cumulative percent release of drugs was estimated using a twin-beam spectrophotometer and spectrophotometric examination of samples at 426 nm ²⁵.

Stability Test: The capacity of a certain formulation in a particular vessel /closure system to stay inside its chemical, physical, microbiological, pharmacological, and toxic parameters during its shelf life is known as the stabilization of a pharmaceutical formulation ²⁶. The storage durability of Nanoemulsion compositions is a serious issue as it is a big problem in the creation of commercially available products. Chosen Nanoemulsion compositions (ME-3) were considered as a high polyethylene bottle and held in plastic cages at a temperature of 40°C and relative humidity of 75 percent. The stability tests were conducted out over a three months period the proportion of medication concentration in the Nanoemulsion composition was tested and verified at frequent intervals ²⁷.

RESULT& DISCUSSION:

Melting Point: The result was founded to be 181°C. The average value was taken from triplicate readings. The reference melting point is about 250 – 251°C.

Solubility: Solubility of curcumin in various solvents was performed by the given method in the methodology section. The obtained data are as follows Table 3.

TABLE 3: SOLUBILITY PROFILE OF LAWSONIA

Standard Curve for Ethanol: Absorbance of different concentrations of curcumin solution was measured at λ max 429 nm. Table 4 shows a calibration curve in Fig. 2.

TABLE 4: STANDARD CURVE DATA

FIG. 2:  STANDARD GRAPH FOR ETHANOL

FTIR Analysis: 1249 cm^-1 owing largely to the overlapped stretch vibration of O=S and carbonyl (C=O) characteristics. Stretch vibrations at 3419cm^-1 owing to O-H groups and high-intensity band 1740 cm^-1 apportioned to blended vibration such as stretch carbonyl bond vibration (C=O), in-plane bending vibrations all over aliphatic CC-C, CC=O, and in bending vibration around aromatic NH stretch of keto and enol setups. All obtained IR data and their graph is given in Table 4 and Fig. 3.

TABLE 4: IR DATA OF LAWSONE

FIG. 3: IR SPECTRA OF LAWSONE

Characterization of Nanoemulsion:

TABLE 5: APPEARANCE OF FORMULATION

FIG. 4: FORMULATED NANOEMULSION

Determination of pH: The obtained PH data of all batches are given in Table 6.

TABLE 6: PH DETERMINATION OF FORMULATED GELS

Viscosity: The viscosity of all batches was checked by Brookfield viscometer at different rpm; obtained viscosity data is given in Table 7.

TABLE 7: VISCOSITY ANALYSIS OF FORMULATIONS

Size of Particle Test: On the basis of the evaluation of all of the five formulated Nanoemulsion gel, two formula F1 and F4 was selected as best Fig. 5, 6.

FIG. 5: PARTICLE SIZE DATA OF F1 BATCH           

FIG. 6: PARTICLE SIZE DATA OF F2 BATCH

Drug Content: Drug content was estimated by the standard curve of Lawsone Table 8. Phase separation test Table 9. In-vitro release Table 10.

TABLE 8: PERCENT DRUG CONTENT OF NANOEMULSION FORMULATION

TABLE 9: PHASE SEPARATION TEST OF ALL SIX FORMULATIONS

TABLE 10: IN-VITRO % DRUG RELEASE OF FORMULATIONS

Antimicrobial analysis Table 11 Fig. 7.

TABLE 11: ANTIMICROBIAL ANALYSIS OF THE DRUG AND FORMULATIONS

FIG. 7: ANTIMICROBIAL SCREENING OF SAMPLES

Stability Data of Developed Formulation: After the 3 month of storage the some parameter was checked and found unchanged during storage Table 12.

TABLE 12: STABILITY DATA OF SOME PARAMETERS

DISCUSSION: Transdermal delivery devices make therapeutic amounts of medication ingredients easier to pass through to the epidermis and into the bloodstream for systemic effects. Topical application of medications having systemic effects has various advantages over other methods, including avoiding The drug's first-pass metabolism in the liver and its associated toxicity, regulating the rate of delivery, and altering drug trafficking in the circulatory system. Two factors are considered while constructing a transdermal delivery system: obtaining enough flux over the skin and minimizing skin permeation lag time. As it promotes epidermal permeability via 2 mechanistic situations: (a) lipids fluidization & (b) lipid stage segregation, Oleic -acid is an epidermal permeability booster candidate. Oleic acid promotes skin permeation by altering the permeability of the stratum corneum. According to the conclusions of the solubility investigation, the drug's thermodynamics activity in the formulations is a primary driving force. Because non–ionic surfactants are often less harmful and cause less skin irritation, they were chosen. The HLB value of a microemulsion of the O/W type. Tween-20 HLB value (16.7), necessary HLB for oleic acid in O/W type emulsion, and HLB value of span-20 (8.6). To increase the porosity profiles, Tween-20, a hydrophilic non-ionic detergent, was utilized to construct these microemulsion complexes. In substitution of propylene glycol, other co-surfactant such as ethanol, butanol and isopropyl alcohol have been used, but propylene glycol was found to have the best clarity and cumulative percent release.

The co-surfactant in microemulsion lowers the surfactant film's interfacial tension leading to a more flexible & kinetic layers structure. The thermodynamically drive power for discharge reflects the drug's comparative activity in different phases. Since medications may be transferred with active ingredients for aqueous dispersion & the extraction solvent to the surface of the epidermis, the comparative activity can be employed to measure the epidermis permeate flux. Furthermore, because surfactants & co-surfactant may be present in each phase, lawsone can be partially dissolved in the exterior phase. The discharge of lawsone form of the active ingredient can replenish the depleted of lawsone from the exterior phase due to penetration into the skin. Oily combinations of  tween-20, oleic acid, and propylene glycol, on the other hand, boosted medication solubility. After comprehensive screening for physical properties and appearance, the final surfactant-cosurfactant ratios were determined.

F-1 through F6 pH values (6, 5.9, 6.12, 6.3, 6.9, 6) units only.pH revealed the smallest variations in all cases. Due to the pH of the epidermis range from 5- 7.0, the pH of the optimized lawsone emulsions formula. It is appropriate for topical and transdermal applications. The produced formulas have steadily grown in thickness (450, 200, 250, 300, 430, 410 cps), as evidenced by the stiffness of the microemulsion system (F1-F6). As anticipated from microemulsion, all specimens displayed Newtonian flow characteristics. Since oil/water microemulsion had greater density than water/oil systems, the stiffness values increased significantly whenever the water content increased or when the system switched to oil/water type. Antimicrobial screening of the drugs and formulations was also screened against bacterial and fungal spices. The optimized microemulsion formula (has particle sizes ranging from 1000-1100 nm with Pdl 0.249-0.290. The data shows that the particle size reduces as the ratio of oil to surfactants: co-surfactants increases. Consequently, microemulsion particles settled close to the body, resulting in a large concentration gradient and improved lawsone absorption.

ACKNOWLEDGEMENT: This research did not receive any specific grant from funding agencies in public, commercial, or non-profit sectors. The author acknowledges the support of Parents and Krishna Kumar in this manuscript.

CONFLICTS OF INTEREST: The author declares no conflict of interest, financial or otherwise.

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    Gupta N, Kumar K, Kumar N and Gupta A: Development and evaluation of laws one (henna) nanoemulsion for antifungal and antibacterial activity. Int J Pharm Sci & Res 2023; 14(2): 952-63. doi: 10.13040/IJPSR.0975-8232.14(2).952-63.

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