Novel anti‐dandruff shampoo incorporated with ketoconazole‐coated zinc oxide nanoparticles using green tea extract

Dandruff caused by Malassezia furfur is a prevailing fungal infection. Although ketoconazole (KTZ) is widely intended for anti‐dandruff treatment, poor solubility, and epidermal permeability limits its use and the marketed KTZ shampoo adversely effects scalp and hair.


| INTRODUC TI ON
Fungal infections have significantly contributed to the rise in diseases and even fatalities for a long time.Infections with fungi could be localized or systemic.Yeasts or dermatophytes bring on the majority of common fungal infections on the skin.Malassezia and Candida species, among yeasts, are common and cause severe skin infections. 1 Dandruff is the most common scalp disorder marked by corneocytes, which cluster together because of their robust and cohesive strength.It appears flaky, with white to yellowish scales, and causes itching.Malassezia species, including, Malassezia restricta, Malassezia sympodialis, Malassezia obtusa, Malassezia sloofiae, Malassezia pachydermatis, and Malassezia furfur are considered the causative agent of dandruff. 2Among them, Malassezia furfur is found to be the most common species. 3South Asia has a 60.1% prevalence of dandruff, compared to a 50% global prevalence. 4Dandruff is most frequently seen between puberty and middle age groups when sebaceous gland activity is at its peak and is more common in men than women because male hormones are probably involved in their production. 5People prone to dandruff may see seasonal variations in their condition, frequently worsening in the winter.The mechanism of dandruff formation is shown in Figure 1. 6,7w available treatments for dandruff include Ketoconazole (KTZ), Zinc Pyrithione, salicylic acid, and Selenium sulfide (Table 1).KTZ (Figure 2) is the most preferred treatment for dandruff due to its effectiveness, less adverse effects, and lower recurrence of dandruff. 8KTZ is an antifungal agent that belongs to the azole (imidazole) class of drugs.It functions as an anti-fungal agent when used topically and orally. 9The FDA has approved the use of KTZ shampoo to treat scalp seborrheic dermatitis and dandruff. 10As KTZ prevents testosterone production, it lowers dihydrotestosterone (DHT), which is used to treat androgenetic alopecia. 11Currently, topical KTZ has been used successfully to treat seborrheic dermatitis. 11Two percent KTZ shampoo is widely marketed.FDA-approved KTZ formulations include Nizoral shampoo, Xolegel gel, and Extina as aerosol foam for treating dandruff and seborrheic dermatitis. 9KTZ-loaded silver nanoparticles, gold nanoparticles, and SLNs have been developed to treat dandruff.The KTZ contained in the shampoo could result in aberrant hair texture, discoloration, irritation, or blister formation on the scalp.Furthermore, it leads to dry or oily hair and scalp. 10Therefore, our formulation aimed to develop an anti-dandruff shampoo with a lower concentration of KTZ up to 1% adjusted by the incorporation of green tea extract thereby minimizing the adverse effects of KTZ and enhancing its treatment effectiveness.In spite of ketoconazole's challenges such as poor solubility, high molecular weight, and restricted epidermal permeability, its therapeutic application remains significant due to its proven effectiveness against specific conditions.Combining ketoconazole with nanoparticles serves to enhance its therapeutic potential which facilitates improved skin penetration.Moreover, the process of nano ionization contributes to the generation of desired polymorphs, characterized by enhanced solubility, dissolution, and permeability.These effectively tackle the concerns associated with ketoconazole's solubility issues, resulting in a more efficacious treatment choice.So, the coating of KTZ onto the surface of nanoparticles can provide better efficacy and drug solubility.It also helps to reduce the amount of KTZ needed in the treatment of dandruff. 12wever, nanoparticles improve the skin penetration of the drug due to their specific functional properties.Nanosization also aids in the production of desired polymorphs with increased solubility, dissolution, and permeability, hence overcoming the solubility issues associated with the chosen medication. 12Shampoos containing herbal extracts are becoming increasingly popular because they are thought to be safer with no reported side effects. 13There is a variety of medicinal herbs that are documented in the literature with proven efficacy to treat dandruff. 14On the basis of these findings, an effort is made to develop an anti-dandruff shampoo using green tea extract (GTE).Green tea is obtained from Camellia sinensis belonging to the family Theaceae. 15It has displayed slower growth of skin cells and gene activation controlling the cell's life cycles.Green tea has been demonstrated to be good for the scalp by Nualsri et al. 16 By controlling the activity of the Caspase-14 protein, which instructs skin cells when to divide and when to stop growing, green tea normalizes the growth cycle of skin cells.Green tea has additionally been demonstrated to soothe skin, lessen irritation, and minimize excessive sebum production. 17,18Green tea is a natural source for the formation of nanoparticles and has a high content of polyphenols.
Epigallocatechin-3-gallate (EGCG), epicatechin, epigallocatechin (EGC), and epicatechin-3-gallate, which act as reducing and capping agents, make up the majority of the compounds in green tea.Sugar, terpenoids, polyphenols, proteins, and alkaloids are plant phytochemicals. 18,19e green synthesis of nanoparticles has become one of the most popular approaches in recent years and has gained major importance.Green synthesis techniques have various benefits, including being easy, affordable, providing good nanoparticle stability, producing non-toxic by-products, and allowing for large-scale production. 20The use of nanotechnology to improve the efficacy of cosmeceuticals is currently flourishing.Compared to conventional medicines, nanoparticulate systems provide focused distribution, painless treatments, tailored therapies, and easier solutions. 21The focus of biomedical research is on zinc oxide (ZnO) nanomaterials.
Large -OH groups are present on the surface of the ZnO nanoparticles, which are easily modified by drugs or other biomolecules and slowly disperse in acidic and strongly basic environments. 22The ZnO nanoparticles are ideal for dermal applications; they primarily remain in the stratum corneum without harmful effects. 23The synthesis of nanoparticles utilizing sustainable, ecologically friendly methods is needed because human civilization already faces a high burden of toxins in all areas of life.The biological method of nanoparticle manufacturing has been tried and used successfully as a safer substitute.
Green synthesis combines a variety of microbial and plant-based extracts to create a mixture of biomolecules.These substances serve as important stabilizers and reductants for the final mediation of nanoparticle production. 24The coating of KTZ onto the surface of nanoparticles can provide greater drug solubility and better efficacy.It also helps to reduce the amount of KTZ needed in the treatment of dandruff.The present study was conducted to prepare ketoconazole-coated ZnO nanoparticles using green tea extract by green synthesis and to evaluate its antifungal activity.
In the context of limited therapeutic options and inherent challenges, including the drawbacks of existing treatment with ketoconazole (KTZ) marked by poor solubility, restricted permeability, and potential drug resistance, we aimed to explore alternative approaches TA B L E 1 Drugs and chemicals used in the treatment of dandruff along with their mechanisms of action.

Zinc pyrithione
Pyrithione, antifungal 0.1%-2.5% Zinc Pyrithione is capable of increasing the amount of copper in the fungal cell, which will reduce the functions of iron-sulphur proteins and depolarize the membrane, which prevents the movement of nutrients and production of energy for enhancing treatment effectiveness while minimizing associated adverse effects by reducing KTZ concentration to 1%. 12 The rising role of nanoparticles is due to improved drug delivery by overcoming conventional limitations. 21The incorporation of nanoparticles, such as ZnO nanoparticles, to enhance drug solubility and targeted delivery likely persuaded this study. 22Moreover, the use of plant extracts such as green tea to synthesize nanoparticles highlights the interest in developing environment friendly methods for medical applications.By assessing the antifungal efficacy and toxicity of ketoconazole coated ZnO nanoparticles synthesized with green tea extract, the study seeks to ascertain the viability of this novel approach in combating fungal infections like dandruff, thus effectively addressing current limitations in dandruff treatment through the integration of nanotechnology.

| Preparation of KTZ-Coated ZnO Nanoparticle
The KTZ-coated ZnO nanoparticles was prepared by a coating method as shown in Figure 4.For the synthesis of nanoparticles, 2.5 mL of 1 M zinc acetate dihydrate, 1 mL of 1 M sodium hydroxide, and 1 mL of 5% GTE was taken in a beaker and stirred for 1 h.
After 1 h, 1% of KTZ was added to the above solution and stirred for 1 h.This solution was then centrifuged at 4000 rpm for 10 min.The supernatant was collected for further analysis.The pellets were washed twice with distilled water and lyophilized for 3 h at 1.2 mbar pressure using Christ Alpha 1-2 LDplus.The nanoparticles were characterized by UV, FTIR, and XRD.

| Characterization of KTZ-coated ZnO nanoparticles
The absorbance of KTZ-coated ZnO nanoparticles was assessed by dispersing the nanoparticle in water with a concentration of 0.   test solution, the effects on CAM, including any hemorrhage, vascular lysis, and coagulation, were noted and recorded over the course of 300 s (5 min).NaOH (1 M) was employed as a positive control and NaCl solution (0.9% w/v) as a negative control. 27

| Preparation of shampoo
Methylcellulose and HPMC were dissolved in distilled water, followed by the addition of sodium lauryl sulphate and coconut oil.
After stirring for a few min benzoic acid, sodium chloride, and EDTA were added.The prepared KTZ-coated ZnO nanoparticles were incorporated into the shampoo and stirred for 30 min.The volume was made up to 10 mL with water.Finally, the pH of the shampoo was adjusted between 6 and 7 by adding sodium hydroxide solution in the desired amount.A few drops of rose oil were added to impart fragrance to the prepared shampoo. 28,29The ingredients incorporated in the shampoo along with its application has been listed in Table 2. 30,31

| Evaluation of shampoo
The formulated shampoo was evaluated for various attributes that included assessments for color, odor, clarity, and texture.To determine its pH, the shampoo was tested at room temperature using pH meter. 32Foaming ability was evaluated using the cylinder shake method, involving the mixing of 1 mL of shampoo with 50 mL of distilled water in a 250 mL graduated cylinder, followed by 10 times shake and measurement of foam volume after 1 min. 33Viscosity was measured using a Brookfield Rheometer.To determine the solid content, 4 g of the shampoo was placed in a clean, dry petri dish, and the liquid portion was evaporated in a hot air oven.The petri dish was then weighed, and the percentage solid content was calculated using the formula given below. 34For dirt dispersion analysis, two drops of shampoo were added to a test tube containing 10 mL of distilled water, followed by the addition of one drop of Indian ink.After 10 times of shaking, the amount of ink in the foam was categorized as none, light, moderate, or heavy.

| Characterization results of KTZ-coated ZnO nanoparticles
The absorbance maxima of KTZ and GTE were observed to be 243 and 209 nm, respectively.UV spectrum of KTZ-coated ZnO nanoparticles in water is shown in Figure 5.The broad peak was observed at a wavelength of 273 nm, which may be assigned to the presence of EGCG in GTE.The spectrum revealed the peak at 365 nm, which is a characteristic feature of ZnO nanoparticles, that may be related to surface plasmon resonance or the movement of electrons from the valence to the conduction band. 24,36 38 and Queiroz et al. 39 In the KTZ-coated ZnO nanoparticles, C=C bond stretching observed at 1558.42 shifted to 1557.33 cm −1 .Alcoholic -OH stretching shifted from 3057.83 to 3116.79 cm −1 , with peaks at 1418.11, 1217.84, and 963.46 cm −1 corresponding to -OH bending, C-N stretching, and C=C bending, respectively.The EE of KTZ in KTZ-coated ZnO nanoparticles was found to be 91.84%.

| Anti-fungal effect of prepared KTZ-coated ZnO nanoparticles
In the preliminary screening, the zone of inhibition was observed only for standard KTZ and KTZ-coated ZnO nanoparticles as shown in Figure 8A.Their respective zone of inhibition is listed in Table 3.
In order to compare the antifungal effectiveness, the study was refined taking similar concentration of standard drug, KTZ. 40,41e zone of inhibition for KTZ (0.161 mg/mL) was not observed, whereas ZnO nanoparticles coated with an equivalent amount of  The study findings correlated with a study carried out by Viswanathan et al. 23 where they prepared a semisolid gel loaded with Ketoconazole-conjugated Zinc oxide nanoparticles and studied its effect on skin disease.The proposed nanoparticles exhibited enhanced penetration and increased retention time on the skin.
Goswami et al. 1 proved that zinc oxide nanoparticles to be robust antifungal agent to eradicate Malassezia growth significantly.V.N et al. 44 revealed that green synthesized Zinc oxide nanoparticles from L. siceraria extract possess potent anti-microbial, anti-dandruff, and antiarthritic activity.

| HET-CAM assay
To determine if the prepared formulation was non-toxic, the cell survival of HCECs was tested after being incubated for 24 h with

| Evaluation of shampoo
The prepared ZnO nanoparticles of KTZ were incorporated in the by the dandruff.In summary, the use of these ingredients together in a shampoo formulation may help to effectively remove dandruff, soothe the scalp, and promote overall hair health. 9,10,12e prepared shampoo as shown in the Figure 9 was opaque, light brown in color, and had pleasant odor due to incorporated rose oil in the formulation.The pH of shampoo was 6.9, which is within the recommended pH range for a shampoo that is 5-7.8. 45e prepared shampoo produced stable foam of 35 mL as shown in Figure 10.In this study, the dirt deposition in the foam wasn't evident as shown in Figure 11, indicating acceptability of the prepared shampoo.The viscosity of the prepared shampoo was observed to be 305.3cp.The prepared shampoo has a solid content of 15.40%.
As a result, shampoo with low solid content is easy to wash off, as shown in

| CON CLUS ION
To our knowledge, this is the first green synthesis-mediated KTZcoated ZnO nanoparticles enriched with GTE.The green nanoparticles enhance the anti-dandruff activity of KTZ and the shampoo improves hair texture, cleansing action while maintaining the pH of scalp.Hence, it can be concluded that KTZ-coated ZnO nanoparticles loaded in a novel shampoo in comparison to marketed anti-dandruff shampoo could be considered as an effective alternate for the treatment of dandruff.
Ergosterol acts as a crucial component of fungal cell membranes.Ketoconazole inhibits the production of Ergosterol by blocking the enzyme cytochrome P450 14-alpha-demethylase, which is necessary for the production of ergosterol from lanosterol 47 Selenium sulfide Keratolytic agent 1%-2.5%It reduces the excessive shedding and itchiness brought on by scalp Fungus and lessens cellular adhesion in the corneum layer.The rate at which thymidine is incorporated into the DNA of epidermal cells is decreased as part of the drug's anti-mitotic mechanism, which reduces the rate of epidermal cell turnover.

F I G U R E 2
Structure of KTZ (Reprinted from Journal of Molecular Lipids, Vol 256, Tawfik A. Saleh, Khaled M.M.Alaqad, Abdur Rahim, Electrochemical sensor for the determination of ketoconazole based on gold nanoparticles modified carbon paste electrode, Page No 40, Copyright (2018), with permission from Elsevier).F I G U R E 3 Chemical structure of the major constituents of GTE: (A) EGCG, and (B) epigallocatechin EGC.(Reprinted from Biochimica et Biophysica (BBA)-Biomembranes, Vol 1863, Stephanie Andrade, Joana Angelica Lourerio, Marica Carmo Pereira, Green tea extractbiomembrane interaction study: The role of its two major components, (−)-epigallocatechin gallate and (−)-epigallocatechin, Page No 2, Copyright (2021) with the permission from Elsevier).
1 wt% and subjecting it to UV spectrophotometric analysis from 200 to 400 nm using Shimadzu UV-1800 Spectrophotometer.The presence of functional groups and possible interaction was detected by the FT-IR spectra of KTZ and KTZ-coated ZnO nanoparticles were obtained using Alpha II compact attenuated total reflectance FT-IR (ATR-FTIR) spectrometer (Bruker, US).The XRD analysis of the pure KTZ and KTZ-coated ZnO nanoparticles were performed using Rigaku X-ray diffractometer (Rigaku Co.).The samples were analyzed at a range of 5-40 2θ with a current of 15 Ma and a voltage of 40 kV.The drug entrapment efficiency (EE) of KTZ in nanoparticles was calculated by an indirect method using the below equation.

2. 2 . 3 | 100 F I G U R E 4
Evaluation of Antifungal effect of prepared KTZ-coated ZnO nanoparticles M. furfur was initially cultured on Sabouraud dextrose agar (SDA) plates overlaid with a few drops of sterile olive oil.One McFarland inoculum was prepared in sterile saline.A modified Malassezia agar medium from Chua et al, 26 was used for evaluating the KTZ formulations.The components of the agar medium per liter were as follows:bacteriological agar 12 g, dextrose 10 g, yeast extract 10 g, peptone 3 g, sodium chloride (NaCl) 2 g, desiccated ox-bile 2 g, L-asparagine 2 g, olive oil 10 mL, and Tween 80 10 mL.The pH of the medium was adjusted to 6.2 using 1 N HCl.The inoculum was spread on the agar plate using a sterile cotton swab.Uniform wells of 6 mm diameter were cut using a sterile cork borer and 50 μL of samples: (A) KTZ, (B) KTZ-coated ZnO nanoparticles), (C) nanoparticles without KTZ (ZnO-GTE nanoparticles) and (D) GTE were deposited in each well for evaluation of its anti-fungal activity.The samples were allowed to diffuse for approximately 3 h at room temperature and then transferred to an incubator set at 35°C.After an incubation period of 3-4 days, the zone sizes were measured using an antibiotic zone scale and results were documented.2.2.4 | HET-CAM assay for toxicity studyHens egg test chick chorioallantoic membrane (HET CAM) experiment was carried out.Using the HET-CAM assay technique as previously published (McKenzie et al., 2015), the toxicity of KTZ-coated ZnO nanoparticles was examined ex vivo or in ovo.White Leghorn chicken eggs that had just been fertilized were obtained from the hatchery and rinsed with tap water to get rid of any dirt or debris.After being candled to remove any faulty eggs, the eggs were incubated for 9 days at 37 ± 0.5°C in a humidified bio incubator.During incubation, the eggs were manually turned three times a day to avoid embryo adhesion to the shell membrane.Eggs were candled on the ninth day of incubation to validate the stage of development by viewing the embryo and CAM blood vessels through the lit shell.The eggs were then maintained upright with the blunt end facing up for 15-20 min at room temperature before being cleaned with 70% ethanol.The shell and inner membrane were removed to reveal the CAM through a small window.On the exposed CAM, test sample solutions (about 300 L) were injected.Following the installation of the % EE = Initial weight of the drug − Weight of the drug in the supernatant Initial weight of the drug × Preparation of KTZ-coated zinc oxide nanoparticles.

3 |
RE SULTS AND D ISCUSS I ONKTZ has been widely used as an anti-fungal agent, especially in the treatment of dandruff caused by Malassezia furfur.In this study, a ZnO nanoparticle of KTZ using green tea extract was prepared by green synthesis and was loaded into shampoo for the treatment of dandruff.The formulated nanoparticles showed enhanced antifungal activity against Malassezia furfur compared to KTZ, and also increased the solubility and permeability of the KTZ.The UV characterization and FT-IR analysis confirmed the formation of nanoparticles, whereas, XRD analysis confirmed the amorphous nature of KTZ in nanoparticles.The pH of shampoo is within recommended range, which improves the quality of hair, strengthens it, and maintains the scalp's ecological balance.The evaluation parameters of the prepared shampoo such as physical appearance, viscosity, foamability, dirt dispersion, and solid content showed ideal results which could enhance patient compliance.
representing its crystallinity.The KTZ-coated ZnO nanoparticles displayed a halo diffractogram confirming the amorphous nature of KTZ as shown in Figure 6.The FTIR spectra of KTZ and KTZ-coated zinc oxide nanoparticles are presented in Figure 7. KTZ showed characteristic peaks of C=H stretching at 3112.09 cm −1 , C=O stretching vibration at 1646.72 cm −1 , 1583.06, and 1513.25 cm −1 corresponding to C=C aromatic symmetric stretching, 1244.28, and 1030.77cm −1 corresponding to C-O stretching of cyclic ether and C-O stretching of aliphatic ether groups, respectively as reported by Ahmed et al (2017)

% 2
Figure 8B.The KTZ-coated ZnO nanoparticles have demonstrated potential to have superior antifungal property when compared with the plain KTZ.The improved potency can be attributed to the increased surface area as KTZ was coated on the surface of the

(
A) a NaCl solution, (B) a NaOH solution, (C) nanoparticles without KTZ (ZnO-GTE nanoparticles), and (D) KTZ-conjugated ZnO nanoparticles.As seen in Figure 10A, CAM was intact when treated with negative control (NaCl solution).Whereas, with positive control (1 M NaOH), blood vessels were disrupted (Figure 10B) and when treated with nanoparticles without KTZ and KTZ-conjugated ZnO nanoparticles, CAM was intact.The black residues seen in Figure 10C,D represents the suspension of blank and formulation.Therefore, no significant cytotoxicity was found in the cells exposed to the nanoparticles.

2
preparation of shampoo.Nanoparticles tend to enhance skin penetration of the drug by increasing its solubility, dissolution, and permeability across the skin surface.The zinc oxide nanoparticles act as carriers for ketoconazole ensuring its sustained release onto the scalp and inhibits the growth of Malassezia furfur.Moreover, the inclusion of Green Tea Extract in the shampoo that contains polyphenols and catechins diminishes scalp inflammation and itching caused F I G U R E 11 Physical appearance of shampoo.TA B L E 5 Results of characterization of nanoparticles loaded shampoo.Foaming ability of the shampoo.