INTRODUCTION:

Plants have several chemical compounds that perform diverse biological roles essential for the resistance towards or amelioration of multiple illnesses or disorders. A person's scalp is protected by hair against elevated temperatures as well ultraviolet light radiation. Furthermore, human hair functions as a crown on the head¹. Consequently, hair requires specialized care to achieve lustre, silkiness, resilience, along with ease of detangling².

The scalp disorder i.e. dandruff is caused by Malassezia fungi, resulting in itchiness along with greasiness^3,4. Dandruff is a prevalent scalp condition impacting over fifty percent of people around the world. Keratinocytes are pivotal in the production and initiation of immune responses during dandruff development. The intensity of dandruff exacerbates throughout winter⁵. Multiple specialty shampoos, including synthetic as well as natural formulations, have been utilized to address dandruff.

Shampoo formulations composed of herbs are cosmetic preparations that utilizes natural plant-derived components^6,7. To enhance the efficacy of shampoo, supplementary chemicals are used with the primary component, surfactant⁸. The primary objective of shampoo formulation is to eliminate filth along with dandruff, while also rendering hair silky as well moisturized⁹.

The objective of this research is to eliminate the reliance on artificial as well chemical-based products offered commercially. Bromelain extract with antidandruff qualities was used, enhancing hair softness and lustre while promoting hair development. Additionally, it has been said that traditional based preparations are cost-effective and not too pricey^10,11.

Bromelain is a proteolytic enzyme extracted from pineapple stems, consisting of a blend of several thiol endopeptidases and other components such as glucosidase, escharase, phosphatase, cellulase, eroxidase, along with many protease inhibitors. It functions by specifically suppressing the manufacture of prostaglandins pro-inflammatory. It is also reported to have analgesic effects¹².

MATERIAL AND METHOD:

Bromelain enzyme extract was acquired from the authorized vendor Herbal Creations in New Delhi. Additional chemicals were acquired from certified suppliers for laboratory grade materials. A complete culture of Malassezia furfur (MTCC: 1374) was acquired through the Institute of Microbial Technology (IMTECH), Chandigarh. The culture was sustained in Emmons modified medium (Hi-Media).

Pre-formulation study

Identification of bromelain extract:

Being an hydrolase enzyme, Crude bromelain possesses proteinaceous nature. A protein analysis, namely the Millon’s as well Ninhydrin tests, was conducted for qualitative evaluation. Furthermore, the shape and color of the Bromelain extract were assessed manually. The substance was subsequently blended with acetone, water, HCl (0.1 N), ethanol as well NaOH (0.1 N), and its capacity to dissolve was assessed.

Bromelain Antifungal Activity:

The antifungal impact was assessed using the disc diffusion technique. A sterilized swab of cotton was used to inoculate Malassezia furfur culture onto the medium plates surface. Disinfected paper filter discs were immersed in pure, bromelain extract 100%, undiluted. Aseptically the discs thereafter were positioned onto plates holding medium, using sterile forceps. Furthermore, discs infused with tea tree oil as well ketoconazole were used as references.

Preparation of Bromelain Shampoo

Accurately measured 2 ml of bromelain extract was transferred to 100 ml beaker and blended using a magnetic stirrer. Methyl cellulose was weighed and then heated with a little amount of water to facilitate its dissolution. Sodium lauryl sulfate and stearic acid were then combined with the aforementioned solution. Sodium metabisulfite was thereafter added and thoroughly mixed with the previously produced API drug solution. The mixture was agitated to achieve uniformity, after which the further needed ingredients—EDTA, PVP, and fragrance—were included and blended. Sodium hydroxide was added to adjust the pH to a near-neutral level suitable for skin compatibility, followed by the addition of water to achieve a total volume of 100 ml. The resulting antidandruff formulation was kept in an appropriate plastic container for further assessment parameters. Table 1 enumerates the numerous substances used in the composition of the current investigation.

Table 1: Ingredients in Shampoo Formulation

S. No.	Ingredients	Quantity	Role
1	Bromelain extract	24 ml	Anti-dandruff/ Anti-fungal / Anti-inflammatory agent
2	Methyl Cellulose	1 gm	Thickener
3	Sodium Lauryl Sulphate	5 gm	Surfactant
4	Stearic Acid	1.4 gm	Emulsifiers/ stabilizer/ lubricants
5	Sodium meta bisulfide	1 gm	Preservative and antioxidant
6	NaOH	0.2 gm	pH Neutralizer
7	EDTA	2 ml	Foaming agent
8	PVP	2 ml	Film former, viscosity controller, and hair fixative
9	Water	q.s. to 100ml	Diluent/vehicle

Evaluation of Bromelain Shampoo

Developed formulation was evaluated for the following parameters^13,14.

Foam volume and its stability test

While foam production is little related to the cleaning efficacy of shampoos, it has significant value for consumers and so serves as a critical factor in shampoo evaluation. The foam volume was evaluated using the blender technique. 40 cc of a 10% shampoo liquid was mixed with regular water for 5 seconds. The height of created foam was measured immediately and after five minutes.

pH test

The pH of shampoos is crucial for boosting hair quality, reducing eye discomfort, and maintaining the natural equilibrium of the scalp. Shampoos devoid of natural extracts have elevated pH levels, whereas those containing natural extracts are acid-balanced, closely aligning with skin pH. The pH of the 10% shampoo preparation in purified water was measured at 25ºC using a pH meter.

Percentage solid content

A high concentration of solids within any given shampoo will hinder its removal or application on scalp and hairs. A clean, dry dish was weighed and combined with 4 grams of shampoo. The shampoo was heated on the hot griddle until the water content dried up. The weight post-drying was determined¹⁵.

Rheological evaluation

Formulation rheology is crucial in determining and regulating several characteristics, including stability over shelf life along with aesthetic qualities such as flow ease upon packaging removal, clarity, application spread ability on hair, as well consistency inside the container. The viscosity profile of any given formulation of shampoo was assessed using Brookfield Synchro-electric RVT viscometer with temperature of 25ºC. The viscosity profile was documented at various revolutions i.e. 0.5, 1, 2.5, 5, 10, 20, 50, and 100 r.p.m.

Stability studies

The stability as well acceptability of the organoleptic qualities (odor, color) of the shampoo formulation over its preservation term demonstrate its physical as well as chemical stability. As per the Bureau of Indian Standards standard, the clear liquid shampoo must be visually inspected to ensure it is devoid of any precipitation. The formulation must exhibit no apparent signs of separation of phases, should be devoid of agglomerated particles, while still maintaining its qualities throughout its shelf life. The shampoo formulation underwent assessment for:

a) Physical stability: The physical stability of the formulation of shampoo was assessed by storing samples at 5ºC, 45ºC, alongside room temperature for a period of 2 months. It was evaluated monthly for changes in pH, appearance, foam height and viscosity of the formulation.

b) Chemical stability: The chemical stability of the shampoo formulation was assessed by comparing the profiles of TLC fingerprint of fresh formulations with that kept for a duration of 2 months. The mobile phase solvent system included toluene and ethyl acetate at a ratio of 9.7:0.3.

Evaluation of shampoo formulation for antifungal activity and its comparison with the antifungal activity of marketed shampoo formulations

The Bromelain containing shampoo formulation prepared as well commercially available shampoos containing the conventional antifungal agents, ketoconazole as well tea tree oil, were assessed for antifungal efficacy against Malassezia furfur using the disc diffusion technique¹⁶. Three duplicates were performed for each samples. The prepared plates were incubated at 32°C, and the zone of inhibition was assessed after 4 days. Reference was kept using filter paper discs saturated with sterile water.

RESULT AND DISCUSSION:

Identification and Physical Characterization of bromelain

Qualitative identification tests indicated that bromelain extract is a protein. The Millon reagent produced a reddish-brown solution. The ninhydrin test is used to identify amino acids and proteins containing free amine groups, resulting in the formation of a purple-blue complex. This outcome is consistent with our prior investigations^17,18. To date, no particular reagent exists that only detects bromelain.

Physical Properties of Bromelain

The extracted bromelain was a powder, exhibiting a yellow to brown hue. It has a subtle pineapple aroma but lacks flavor. It was determined to be soluble in water at a pH of 6 to 7. The solid content constituted 33%. The many physical traits noted are listed in Table 2 below.

Antimicrobial Activity of bromelain extract

The antifungal efficacy against Malassezia furfur was assessed using the disc diffusion technique. The Zone of Inhibition (ZOI) shown by the experimental sample was compared with the conventional antifungal drugs, ketoconazole as well as the Tea tree essential oil i.e. Melaleuca alternifolia, against Malassezia furfur, as presented in Table 2. Rahmi et al. (2019) also observed a comparable antifungal activity of bromelain extract against Streptococcus mutans¹⁹ as did López‐García et. al. in 2012 against Fusarium species²⁰.

Table 2: Zone of Inhibition (ZOI) against Malassezia furfur

S .No	Drugs	ZOI in mm (Mean ± SD)
1.	Bromelain extract	13.5 ± 0.31 *******
6.	Tea tree oil	16.5 ± 0.42 *******
7.	Ketoconazole	19.5 ± 0.16***

***p value < 0.01

Evaluation of Shampoo

Foam Volume and Stability test

The test sample exhibited favorable foaming properties, with a foam volume of 180±0.01ml immediately after agitation. The volume is 160.2 ± 0.45 ml after standing for 3 minutes.

Rheology study results

The viscosity profile of the shampoo was assessed using a Brookfield viscometer at various revolutions per minute (rpm). The findings are shown in Table 3 and Figure 1. The findings demonstrated that the shampoo exhibited pseudoplastic behavior, a desirable characteristic in shampoo composition²¹. At low RPM, these shampoos exhibited increased viscosity. As shear increased, viscosity decreased, facilitating easier application into the hair.

Table 3: Viscosity profile of shampoo formulation at different rpms

S. No.	Rotation per minute	Viscosity Observed
1	0.5	29260
2	1	27020
3	2.5	24113
4	5	21780
5	10	19500
6	20	16800
7	50	14440
8	100	12500

Fig. 1: Graph showing pseudo plastic behavior of shampoo (viscosity decreases with increase in rpms)

Physical stability

The stability investigations outcomes for the shampoo composition are provided in Table 4. The stability investigations of the shampoo composition revealed no significant changes in foam formation, detergency, pH, or viscosity during the testing period. The stability along with acceptability of the organoleptic qualities (color, appearance, odor) of the shampoo preparation over the period of storage suggested that the prepared shampoo was physically stable.

Table 4: Stability studies of the antidandruff shampoo formulation

Parameter	1 month	2 month
Physical appearance	No observed change	No observed change
Colour and Odour	No observed change	No observed change
Foam ability as well stability in distilled water	179 ± 1.33	178 ± 1.02
Surface tension	33.0 179 ± 1.47	32.3 179 ± 0.73
Detergency (%)	76.02 ± 0.01	75.25 ± 0.04
Viscosity (at 50 rpm)	14,200± 1.223	14,330±0.111
pH	5.6 ± 0.05	5.6 ± 0.12

Chemical stability

The profile of TLC fingerprint of the fresh formulation of shampoo alongside the profile of the formulation kept for two months, were found to be similar. The Rf values of several bands in both formulations were almost identical, ranging from 0.13 to 0.97 units under 254 nm UV light and daylight.

This demonstrated that there was no deterioration of any components, as seen in the fresh formulation, indicating a stable preparation with constituents exhibiting acceptable compatibility.

Antifungal effect of formulated shampoo against Malassezia furfur along with comparison with market preparations

The developed antidandruff shampoo was assessed for antifungal efficacy against Malassezia furfur using the disc diffusion technique. The zone of inhibition (ZOI) of the experimental the preparation was compared to the antifungal efficacy of a commercial synthetic antidandruff agent ketoconazole as well as herbal antidandruff agent i.e. Tea tree essential oil (Melaleuca alternifolia), against Malassezia furfur. The antifungal efficacy of the shampoos examined was ranked as follows: ketoconazole > prepared shampoo > tea tree oil, as seen in Table 5 and Figure 2.

Table 5: Anti-fungal activity of formulated and marketed anti-dandruff shampoos

S.No	Anti-dandruff Shampoo	Zone of Inhibition (in mm) Mean ± SD	P value
1.	Bromelain containing	14.1 ± 0.28	0.0106
2.	With ketoconazole	15.0 ± 0.20	0.012
3.	With Tea tree oil	13.6 ± 3.46	0.022

Fig. 2: Comparative representation of antifungal activity of formulated and marketed antidandruff shampoos

The developed antidandruff shampoo had a lesser antifungal impact compared to the commercially available antidandruff shampoo with synthetic components, although surpassed the marketed shampoo with herbal constituents. The disparity in antifungal efficacy shown by the shampoo developed in the current investigation compared to the ketoconazole formulation was not significant. Consequently, our formulation incorporating Bromelain extract shown efficacy against the fungus Malassezia furfur.

CONCLUSION:

Scalp dandruff is a prevalent issue in cosmetic dermatology. Malassezia furfur is the primary pathogen responsible for dandruff, and its lipase activity generates pro-inflammatory free fatty acids, leading to cutaneous irritation and tissue damage. Among the several formulas available for dandruff treatment, antidandruff shampoos are the most prevalent. The current research used pineapple stem extract, which contains bromelain, as an anti-inflammatory and anti-dandruff ingredient in the formulation of herbal shampoo. The antifungal activity test revealed a zone of inhibition of 14.1 ± 0.28 for bromelain-containing shampoo, compared to 15.0 ± 0.20 for ketoconazole and 13.6 ± 3.46 for tea tree oil formulations currently available, suggesting bromelain's potential efficacy in treating dandruff. The designed shampoo was determined to exhibit all the essential characteristics required of a quality shampoo. Consequently, it may be stated that the preparation was successful, creating new opportunities for herbal combinations in hair care systems.

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Received on 03.12.2024 Revised on 22.01.2025

Accepted on 17.03.2025 Published on 28.03.2025

Available online from April 01, 2025

Research J. Topical and Cosmetic Sci. 2025; 16(1):1-5.

DOI: 10.52711/2321-5844.2025.00001

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.