Dandruff can be classified as disorders of the sebaceous gland or skin scaling disorders. 

Dandruff can be of two types, they are: 

The fungi Malessezia can lead to dandruff by either or both of the following mechanisms-

Dandruff is an overall scalp disorder/disease. The treatment of dandruff includes application of topical, antifungal, or other products. Some of the courses of treatment using therapeutic mechanisms include the following in the diagram (Figure 1). ⁶ 

Let us look at the target area of treatment by analyzing the main cause of dandruff and how to treat them-

Since there are enough synthetic anti dandruff products in the market such as salicylic acid, ketoconazole, coal tar and many more of them, one of the drawbacks of these synthetic products is that they have some side effects after prolonged use. So, in order to reduce these side effects, we as researchers are trying to find out natural alternatives that can help treat dandruff naturally. 

Our aim is to prepare a medicated hair care product which could be easily applicable as well as it can be used on daily purpose even in their busy schedule. The idea of formulating the herbal anti-dandruff hair mask is to soothe the irritating, oily and flaky scalp by controlling dandruff thus giving you itchy free healthy scalp and nourished hair. So, in order to increase the contact time of the product with customers' usage, we also need to focus on the formulation of the product which will deliver the therapeutic effect even after less amount of contact time. The product should also be able to release the drug into the system efficiently in a short period of time as well. Our route of administration is topically or through the skin surface. So, we can use one of the aspects of formulating the product which includes the above criteria i.e. an Emulgel.

Preparation of an ideal Emulgel is illustrated in the Supplementary Fig S4.

Herbs such as Moringa leaves (Moringa oleifera), Mexican mint leaves (Coleus amboinicus) and Hibiscus leaves (Hibiscus rosa-sinensis) were chosen carefully for testing their antifungal properties against dandruff-causing species ^{7, 8, 9, 10}. 

The following three herbs were firstly extracted with various solvents such as DW, Ethanol, and results showed that most of the yield was extracted better with water compared to ethanol extracts. But ethanolic extracts were preferred over aqueous extract due to its enhanced activity.

Later, an antifungal bioassay was performed using these extracts on culture Malessezia species to see whether the herbal extracts were able to inhibit the growth of organisms individually as well as combined where Mexican mint was found to be most effective against the species. So, Mexican Mint was further considered for future studies.

Pumpkin seed oil, Tween 80, Stearic acid, Mexican mint extract, Glycerine, Citric acid, Carbopol 940, Triethanolamine, Distilled water and Perfumery agents.

The leaves were collected from nearby surrounding garden areas with optimum climate conditions and dried under artificial drying conditions using a hot air oven at 70 degrees for 7.5 hours ¹³. Then the dried leaves were powdered in a grinder and later a weighted quantity of powder was mixed along with ethanol (say 10 grams/100ml), and it was extracted using condenser for 8-10 hours. The extract was later collected by filtering the filtrate and drying it to consistent extract using a water bath.

1. Extractive values 21

Extractive values were performed in order to know which extract would give most of the plant soluble extraction in that particular solvent. So, about 50 ml of solvents like DW & Ethanol and 2.5 gm of dried herb extract were taken in a conical flask and continuous stirring by magnetic stirrer was provided for 6 hours and left for maceration for 18 hours. Then these solutions were filtered out and 12.5 ml of the respective filtrate was transferred into a pre weighed porcelain dish and the solvents were evaporated in a water bath according to the required temperatures. After evaporation, these extracts were placed in a desiccator and readings were taken until constant weights were achieved. Following formula was applied for extractive values:

2. Antifungal Bioassay 8, 9, 10

Well Diffusion method was used wherein a mixture of Brain Heart Infusion broth (BHI) & Sabouraud Dextrose Agar (SDA) media were poured into sterile plates and solidified, the cultures were inoculated using swab & wells were created using cork borer. Into these wells, the standard & test samples were added using micropipettes & the plates were incubated for 24 hours outside the incubator and results were noted. 

In order to conduct this assay, primary identification and determination of species Malessezia furfur (MTCC NO 1374) were done using the gram staining method which is shown in Supplementary Fig S5 and Supplementary Table S1.

This assay method was performed in order to conclude the activity of selected herbal extracts against Malessezia furfur individually and combined.

3. Absorption Maxima (λmax)

The absorption maxima were performed to determine the optimum wavelength at which maximum energy or light is absorbed by the active constituents. 

For determination of the spectra, PBS solution of pH 7.4 was prepared according to the method described in I.P 2022 and pH was adjusted to 7.4 using digital pH meter before any quantitative estimation ²². Similarly, another solution of PBS was prepared using the above procedure and adjusted to pH 5.5 ²². This step is mandatory as we will determine in-vitro drug diffusion studies under pH 7.4 and 5.5 which are pH values for blood and scalp respectively. When the pH of the solution changes, there is ionization in some of the molecules of the solution, thus the structure of the molecule changes, which will affect the determination of absorbance. Hence absorption maxima will be measured in two different pH systems ²³.

Mexican mint extract of accurately weighed amount (100mg) were dissolved in ethanol and then made up to 10 ml with PBS of each pH. Each ml of the stock solution contains 10 mg of extract containing active constituents (primary stock solution). From this primary stock solution, 1 ml was withdrawn and made up to 10 ml with PBS of each pH (secondary stock solution) was taken in standard cuvette and scanned in the range of 200-700 nm in a UV spectrophotometer. Therefore, further all measurements were taken according to requirements of the test.

4. Standard curve for drug diffusion studies and quantitative tests

A) For Drug Diffusion studies and drug content - Mexican mint of accurately weighed amount of extract (0.5 gm) was dissolved in 1 ml ethanol which becomes the primary stock solution. From this primary stock solution, concentrations of 30, 60, 90, 120 and 150 mg were prepared and made up to 1 ml with PBS of each pH (secondary stock solution). From this stock solution, further 100 times dilution was done such as each test tube contains 10 ml for reading and was scanned at 672 and 677 nm in a Ultraviolet-Visible (UV) spectrophotometer.

B) For Phenol Content24 - A set of standard solutions of gallic acid (20, 40, 40, 60, 80 and 100 μg/ml) were prepared from a stock solution of 0.01 gm/10 ml with each ml containing 10 mg of GAE. The Folin-Ciocalteu assay method was used for the determination of the total phenol content. The reaction mixture consists of 1 ml of prepared reading and 9 ml of distilled water taken in a volumetric flask (25ml). One milliliter of Folin-Ciocalteu phenol reagent was treated to the mixture and shaken well. After 5 minutes, 10 ml of 7 % Sodium carbonate (Na₂CO₃) solution was treated to the mixture. The volume was made up to 25 ml. Incubated for 90 min at room temperature and 10 times dilutions were prepared. Record the standard solutions against the reagent blank at 550 nm with an Ultraviolet-Visible (UV) spectrophotometer.

C) For Alkaloid Content24 - A set of reference standard solutions of atropine (20, 40, 60, 80 and 100 μg/ml) were prepared from a stock solution of 0.01 gm/10 ml with each ml containing 10 mg of atropine. 5 ml of bromocresol green solution and 5 ml of phosphate buffer (pH 4.7) were added in the above prepared solutions and were transferred to a separating funnel and yellowish chloroform layer collected in a 10-ml volumetric flask and diluted to the volume with chloroform. Further, 10-time dilution was done. The absorbance was determined against the blank reagent at 470 nm with an UV-Visible spectrophotometer.

5. TLC/Thin Layer chromatography

Thin layer chromatography (TLC) is an affinity-based method used to separate compounds in a mixture. So, following procedures were performed.

Mobile phases were prepared according to the requirements and kept in the saturation chamber (beaker/measuring cylinder) for saturation. Meanwhile, spots of respective samples were spotted onto the stationary phase and left for a minute to dry off. After which, these plates were carefully inserted in the chamber and left for development of spots. Further derivatization of these spots was done using iodine vapors. Spots were visualized in TLC UV chamber and calculation of Retardation factor was done by using the formula-

Retardationfactor=Distance travelled by the compoundDistance travelled by the solvent front

6. Chemical tests (Qualitative Analysis)

The alcoholic extract of Mexican mint was subjected to following preliminary phytochemical analysis:

7. Quantitative Analysis 24

This analysis is done to quantify the presence of chemical constituents in the herbal extract. The procedure for standard curves is mentioned in point 3 of Preformulation studies while for sample preparation and estimation, similar procedure was followed as mentioned below- 

A) Phenolic content- The reaction mixture consists of 1 ml of prepared sample (1gm/ml) and 9 ml of distilled water taken in a volumetric flask (25ml). One millilitre of Folin-Ciocalteu phenol reagent was treated to the mixture and shaken well. After 5 minutes, 10 ml of 7 % Sodium carbonate (Na₂CO₃) solution was treated to the mixture. The volume was made up to 25 ml. Incubated for 90 min at room temperature and 10 times dilutions were prepared. Record the standard solutions against the reagent blank at 550 nm with an Ultraviolet (UV) or Visible spectrophotometer. The concentrations were calculated using the standard graph plotted above. 

B) Alkaloid content- Samples were initially extracted with 2N HCL and chloroform and separated the chloroform layer. 5 ml of bromocresol green solution and 5 ml of phosphate buffer (pH 4.7) were added in the sample solution (0.5 gm/ml) and were transferred to a separating funnel and yellowish chloroform layer collected in a 10-ml volumetric flask and diluted to the volume with chloroform. Further 10-time dilution was done. The absorbance was determined against the reagent blank at 470 nm with an UV/Visible spectrophotometer. The concentrations were calculated using the standard graph plotted above.

C) Flavonoid content- The reaction mixture consists of 1 ml of sample solution (0.5gm/ml) preparation and 4 ml of distilled water was taken in a 10 ml volumetric flask. To the flask, 0.30 ml of 5 % sodium nitrite was treated and after 5 minutes, 0.3 ml of 10 % aluminum chloride was mixed. After 5 minutes, 2 ml of 1M Sodium hydroxide was treated and diluted to 10 ml with distilled water. Further 100-time dilution was done. The absorbance for standard solutions was determined against the reagent blank at 510 nm with an UV/Visible spectrophotometer. The concentrations were calculated using the standard graph.

8. Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC)

Minimum Inhibitory concentration (MIC) of fungicide is the minimum concentration required to inhibit/arrest the growth of all microorganisms in the culture and Minimum fungicidal concentration (MFC) is the minimum concentration required for the complete death of fungal strains present in the culture. This test was done to determine the exact strength required to be used in the effective formulation of the mask.

Materials: Saline solution (0.9% w/v), nichrome loop, plugged test tubes, Petri plates, nonabsorbent cotton, micropipette tips, beaker, conical flask, test solution, solvents like DW and alcohol, disinfectant, SDA Broth, BHI Broth, agar-agar, inoculum of culture, Mexican Mint extract.

9. Anti- Inflammatory Assay 26

The principle objective behind the Egg Albumin Denaturation Assay is to determine whether compounds can stop or hinder egg albumin from becoming denatured under particular circumstances. Denaturation is the term used to describe how a protein changes in structure and loses its biological activity. The egg albumin denaturation assay is based on the idea that substances with anti-inflammatory qualities may be able to stabilize protein structures and prevent denaturation, which is frequently linked to inflammation and tissue damage. It measures a drug or compound’s capacity to prevent or lessen egg albumin denaturation to evaluate its anti-inflammatory effects. Mexican mint is an anti-inflammatory herb due to its thymol content present in the herb.

Preparation of 1% egg Albumin: 1 gm of egg albumin powder was dissolved in 100 ml of DW and mixed slowly to avoid any lump formation. DW should not be warm otherwise egg albumin may coagulate. Filter through the cotton filter into a beaker.

Prepare a stock solution of Mexican mint alcoholic extract of 0.1 gm/100 ml alcohol. From this, solutions were further diluted for readings in range from 50,100,150,200 & 500 ug with alcohol and made up to 2 ml each for following set of each reading- 

Similarly, a reading of standard diclofenac solution (500 ug/ml) was prepared by following the above procedure.

These solutions were incubated at 37°C for 30 mins and later heated in a water bath for 15 mins at 70°C. Readings were taken at absorbance 600 nm.

% inhibition can be calculated by the formula- 

% inhibition=Absorbance of control-Absorbance of Test-Absorbance of BlankAbsorbanceof Control×100

Homogeneity 4

It can be defined as a formulation which has its smooth consistency, no coarse particles and has uniform color. The product was taken in a beaker and was observed for these properties and results were noted.

2. Spreadability 27, 4

The spreadability of the product was measured by spreading 0.5 g of the gel on a glass plate and then a second glass plate was employed. 100 gm of weight was permitted to rest on the upper glass plate for 5 min. The diameter of the circle after spreading of the Emulgel was determined by formula-

                                           S = M*L/T

Where S = spreadability, M = Weight tied to upper slide, L = Length of glass slides, T = Time required to separate the slides.

3. Viscosity 4

100 gm of product were taken in 250 ml beaker. The prepared solution is placed in the Brookfield Viscometer and a proper speed is selected for the spindle according to the expected viscosity of the sample. The Brookfield Viscometer determines viscosity by measuring the force to turn the spindle in the solution at a given rate.

4. In vitro diffusion studies ⁴

The objective of in vitro dissolution testing is to evaluate the variables that affect the rate and extent of release of a drug substance from the finished dosage form, and in turn, the in vivo performance of the drug product. Franz cell diffusion apparatus was used for 6 hours study while parchment membrane/cellulose membrane was used as a passing membrane. Prepare PBS solution of pH 5.5 and 7.4 according to the procedure in IP 2022 ²². At an interval of 30 minutes, take the reading of 2 ml with appropriate measures and take the absorbance at 672 and 677 nm for pH 5.5 & 7.4 respectively.

5. Drug content 4

This test was done in order to determine the strength of API in the product. So, 0.05 gm formulation was mixed in 25 ml of PBS buffer of pH 7.4 on a conical flask, then kept stirring using a magnetic stirrer for about 2 hrs and occasionally shook the flask with mild action. Record absorbance at 677 nm and calculate the drug content using standard curve equation.

6. Anti-fungal bioassay of product

The antifungal efficacy of both the products in two conditions were determined-

A) Storage under normal conditions (25°C).

B) Storage at higher temperature (45°C).

Similar procedures as mentioned in Preformulation studies- point 7C was followed and zones of inhibitions were noted after 24 hrs.

7. Stability studies

Both the products were stored at accelerated stability studies conditions (45°C, 60% RH) for 30 days and were observed after each 10 days to determine its consistency, its appearance, color and presence or absence of phase separation were noted.

It was found to be 5.2 % and 4% for water and ethanol extracts respectively. Further, only ethanol extracts were taken into consideration due to degradation of water extracts after some time and presence of key activity. Refer to Supplementary Table S2 for exact values.

2. Antifungal Bioassay

Refer to Supplementary Fig S6 for images.

Following observations were made

3. Absorption maxima (λmax)

Following graphs were noted for extracts (See Supplementary Fig S7)-

At pH 5.5, it was found to be 677 nm and at pH 7.4, it was found to be 672 nm.

4. Standard curve for drug diffusion studies and quantitative tests:

Refer to Supplementary Fig. S8, S9 and S10 for graphical interpretations.

5. TLC/Thin Layer Chromatography

It was found that thymol (0.74) was present and identified in the Mexican Mint. Refer to Supplementary Fig. S11 and Table S3 for more.

6. Chemical test (Qualitative analysis)

Refer to Supplementary Fig. S11 and Table S4 for more.

7. Quantitative analysis- Flavonoid content, Phenolic content and alkaloid content 24

In qualitative estimation, it was found that there was no detection of alkaloids because of which it was also not detected while performing alkaloid content test for the herb. However, Liquid chromatography - mass spectrometry (LC-MS) can be used as a possible method for evaluation of alkaloids of this herb. Refer to Supplementary Table S5 for values.

8. Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC)

When calculated for MIC/MFC ratio, it was found to have Fungicidal action with an index of 2 ²⁵. Thus, Mexican Mint alone can be used for treating dandruff which is harsh and causes inflammation of scalp due to dandruff as this herb also shows anti-inflammatory action in further tests. Refer to Supplementary Fig S13 and Table S6 for more.

9. Anti- Inflammatory Assay

While comparing our sample (Mexican mint) with standard (Diclofenac) we found that at the same concentration (500 ug/ml) where standard shows 95% inhibition, the sample shows 53.12% inhibition. But at lower concentration i.e. 100 ug/ml, it was found to show 99.3% inhibition. Refer to Supplementary Fig S14 for image.