Powder shampoo formulation with antioxidant activity: in vitro perspectives

Hair acts as a defense barrier against external factors (Erdoğan, 2017). This is particularly true in the case of scalp hair offering protection against UV radiation (Zaidi & Lanigan, 2010). Despite vital role of scalp hair as defensive barrier and its contribution to overall physical appearance, it is believed that hair care is not taken as seriously as skin care (Trüeb, 2021). Hair shaft majorly consist of cortex, cuticle cells and medullary cells, whereas hair follicle is essential for the generation of hair. The hair follicle follows a pattern of continuous growth and rest cycle (Erdoğan, 2017), which are shown to be influenced by various internal and external factors such as endocrine, neural stimuli, vascular, age, nutritional habits and maintenance of personal hygiene (Trüeb, 2021). The effects of pollutants, dust, chemicals and ultraviolet radiation exposure onto the scalp leads to hair damage via the generation of reactive oxygen species. Reactive oxygen species are a term used to describe highly reactive intermediates produced in cells by various mechanisms (Hoting & Zimmermann, 1996; Nogueira, Dicelio, & Joekes, 2006; Signori, 2004). These intermediates, when not sufficiently countered by antioxidant defenses, could cause oxidative damage to cellular components and such damages have been implicated in the etiology of many disorders affecting key organs such as cardiovascular system, kidney and nervous system (Bandyopadhyay, Das, & Banerjee, 1999; Liguori et al., 2018). Therefore, it is not surprising that oxidative stress has been studied as possible mediators of dyshomeostasis associated with hair health. Upton et al. showed that dermal papilla cells (DPC) from balding men are more sensitive to oxygen and secrete higher levels of negative hair growth regulators in response to hydrogen peroxide than occipital DPCs. Further, DPCs from balding men were reported to be less adept at handling oxidative stress in spite of higher levels of catalase and glutathione levels (Upton et al., 2015). Androgenetic alopecia patients have been reported to be associated with lower levels of superoxide dismutase and higher levels of lipid peroxidation in plasma along with reduced total antioxidant capacity (TAC) (Prie, Iosif, Tivig, Stoian, & Giurcaneanu, 2016). 8-hydroxy deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, and C-reactive peptide are reported to be higher in serum of patients afflicted with alopecia areata (Mustafa, Khashaba, Fawzy, Baghdady, & Rezk, 2021). Acharya and Mathur in their review provide an account of investigations that suggest that alopecia areata is associated with oxidative stress, and magnitude of oxidative stress correlates with severity of alopecia areata (Acharya & Mathur, 2020). Peterle et al. suggest that malondialdehyde and advanced glycated end products could serve as biomarkers for alopecia areata (Acharya & Mathur, 2020).

Shampoos are cosmetic preparations used in cleansing hair and scalp as most of the contaminants are water insoluble thereby requiring efficient surfactants to remove those (Robbins, 1998). Water-based shampoos have been very prevalent in the market space since a long time. One of the primary and vital concerns over the use of liquid shampoo is the utilization of plastic bottles and sachets for the storage. Macroplastics have been well recognized as contaminants in aquatic domain and are known to cause tremendous physical injuries and even death due to either entanglement or ingestion (Cole, Lindeque, Halsband, & Galloway, 2011; Derraik, 2002; Ryan, 1987, 2008). There are concerns over breakdown of macroplastics into microplastics and effects of the latter on ecosystem. It suggest that they can act as a possible threat to the ecosystem as these small plastic fragments were found in the digestive system of birds, crustaceans, turtles, seabirds and fish (Cole et al., 2011; Derraik, 2002; Ryan, 1987, 2008). Accumulation of such small fragments plastic in the gut further leads to starvation, malnourishment and premature death of the affected species (Boerger, Lattin, Moore, & Moore, 2010; Ryan, 2008). Therefore, it is highly desirable to use biodegradable materials in packing and distribution of personal care products. Furthermore, certain chemicals used in market available shampoo such as zinc pyrithione, selenium sulfide are known to cause damage to the keratinocytes (Gilbertson, Jarrett, Bayliss, & Berk, 2012; Lamore, Cabello, & Wondrak, 2010). These could also cause damage at molecular level thereby could be potentially genotoxic in nature (Lamore et al., 2010). Because of the abovementioned reasons, herbal shampoo is becoming widely acceptable due to the incorporation of plant-derived substances (Godeto et al., 2023; Saripalla et al., 2021). The secondary metabolites of plant extracts are known to be enriched with vitamin C, vitamin E, vitamin B, polyphenols, flavonoids, tannins, carotenoids and organosulfur compounds (Godeto et al., 2023; Sharifi-Rad et al., 2020), which serve to counter the effects of pro-oxidants, thereby preventing pathologies associated with oxidative stress (Godeto et al., 2023; Saripalla et al., 2021; Sharifi-Rad et al., 2020) as well as aiding in improving hair strength, hair texture, volume and hair growth. Usage of herbs such as aloe vera (Chaitanya & Jaiswal, 2022), Acacia concina (shikakai) (Saripalla et al., 2021), Azadiracta indica (Neem) (Chaitanya & Jaiswal, 2022), Cyclea peltata (raj patha) (Cheni Cheri, Kizhakke Veettil, Pradeep, & Nayak, 2022), Ocimum temple (tulsi) and Phyllanthus emblica (amla) (Saripalla et al., 2021) has proven to be highly beneficial not only as surfactant but also resolving several scalp concerns such as dandruff, seborrhea dermatitis in the shampoo formulation (Chaitanya & Jaiswal, 2022; Cheni Cheri et al., 2022; Saripalla et al., 2021). It could be argued that hair cleansing formulation containing surfactants and functional ingredients in dry or powder format offer the advantage of reduced cost of manufacturing and storage in environmentally friendly containers.

Given the need for powder shampoos and fictionalization of shampoos with antioxidant attributes, the present study developed a powder shampoo formulation containing Cinnamomum zeylanicum bark extract, alpha olefin sulfonate (AOS) and sodium cocoyl isethionate (SCI) for evaluation of detergency action and antioxidant potential in vitro.

Ammonium molybdate, ascorbic acid, disodium hydrogen phosphate, FC reagent, gallic acid, hexane, methanol and sodium bicarbonate were obtained from Sisco Research Laboratories (India). AOS was procured from BRM Chemicals, Rajasthan, India. Mannitol and maltodextrin were procured from Bioven Ingredients Pvt. Ltd., UP, India. Microcrystalline cellulose was from Accent Microcell Pvt. Ltd., Gujrat, India. SCI was from Pratha, India. Cinnamon bark was procured from local market. All other chemicals used in the present study were of analytical grade.

Cinnamomum zeylanicum bark powder was extracted twice with methanol at room temperature. Supernatants obtained by filtration were pooled and concentrated under vacuum at 45^oC to obtain a pleasant-smelling dark-colored viscous oleoresin (Mathew & Abraham, 2006).

Powder shampoo containing extract of bark of Cinnamomum zeylanicum (DSCE) was prepared by mixing AOS, SCI, microcrystalline cellulose mannitol, carboxymethyl cellulose, maltodextrin, methanolic extract of bark of Cinnamomum zeylanicum and sodium benzoate. The details regarding the composition are given in Table 1. Above described ingredients were mechanically blended to obtain a homogenous powder. For comparison of antioxidant potential, a formulation designated as “DS” was prepared by replacing the methanolic extract of bark of Cinnamomum zeylanicum with maltodextrin.

For evaluation of antioxidant potential in vitro, dry shampoo containing extract of bark of Cinnamomum zeylanicum (DSCE) and dry shampoo without extract of bark of Cinnamomum zeylanicum (DS) were extracted with methanol, and the methanol soluble fraction was tested for radical scavenging activity, total phenolic content and TAC as described below. The cinnamon extracted was dissolved in methanol for evaluation of antioxidant potential in vitro.

Folin-Ciocalteu method was used for the estimation of the phenolic content in of the dry shampoo formulations. The phenolics in the extract reduce the reagent to produce molybdenum–tungsten blue, which is read at 765 nm, intensity of the color produced was found to be in linear relationship with the concentration of phenolics. The experiment was carried out in 96 well plate. The assay was initiated by the addition of 85µl of distilled water followed by 10µl of sample (methanolic extracts) and 25µl of FC reagent (1:1 dilution). Post an incubation period of 6 minutes, 100µl Na₂CO₃ (75g/l) was added and palate was in dark for 90 min. The plates were read at 765 nm using a microplate reader. Gallic acid was used as standard and results were expressed as gallic acid equivalents (Herald, Gadgil, & Tilley, 2012).

The TAC of the test extract was spectroscopically determined using phosphomolybdenum method, which is based on the reduction of Mo (VI) to Mo (V) complex under acidic conditions. 50µl of the samples was mixed with 500µl of the reagent (28 mM sodium phosphate, 4 mM ammonium molybdate and 0.6M sulfuric acid) and kept in boiling water bath for 90 mins. After cooling to room temperature, the absorbance was measured at 695 nm. Ascorbic acid was used to obtain the calibration curve (Alam, Bristi, & Rafiquzzaman, 2013).

The radical scavenging activity exhibited by dry shampoo formulations was determined by monitoring scavenging of the free radical α, α-diphenyl-β-picrylhydrazyl (DPPH) spectrophotometrically. 100µl of DPPH reagent (8mg in 100 ml methanol) was incubated along with 100 µl of sample (ascorbic acid or dry shampoo formulations or the bark extract) in 96 well plate for 30 min in dark and read at 514 nm (Lee, Oh, Cho, & Ma, 2015).

The detergency action of dry shampoo formulations was evaluated by the Barnet and Powers test with modifications (Patidar, 2018). Briefly 1 g of wool yarn was greased with vegetable oil and weighed. The greased wool yarn samples were then placed in 200 ml of water suspension prepared with one gram of dry formulations. The flasks were shaken for four minutes at 35 degree Celsius. The wool yarn samples were then gently washed with water and dried. Weights of the wool yarns were recorded to determine the detergency and cleaning action attributable to emulsification property of the formulation.

Statistical Analysis: Data have been represented as Mean ± SD (n = 3). ANOVA and Tukey post hoc tests were determined to determine the significances in differences of means.

Antioxidant activity of methanolic extract of bark of Cinnamomum zeylanicum

Antioxidant activity of the cinnamon extract is depicted in Table 2. As depicted in the table, the extract possessed appreciable amounts of phenolic content and exhibited strong TAC and radical scavenging activity.

In vitro antioxidant attributes of the shampoo formulations

As depicted in Figure 1A, DS (dry shampoo without cinnamon extract) possessed negligible amounts of phenolics, while DSCE (dry shampoo with cinnamon extract) contained significantly higher amounts of total phenolics. Addition of cinnamon extract to shampoo formulation caused increase in TAC by several folds (Figure 1B). The data on radical scavenging activity of shampoo formulations are Figure 2A. DS (dry shampoo without cinnamon extract) possessed negligible DPPH scavenging activity, while DSCE (dry shampoo with cinnamon extract) was capable of significantly higher DPPH scavenging (>70%). Ascorbic acid was capable of strong DPPH scavenging activity with an EC50 value of 14.0 ± 0.71 μg/mL (data not shown). As depicted in Figure 2B, DSCE and DS were capable of removing more than 95% of oil while water washing (WS) was associated with removal of small amounts of oil (17%). Incorporation of cinnamon bark extract did not affect the detergency efficacy of the dry shampoo formula.

The present study aimed at developing a unique blend of powder shampoo formulation containing cinnamon bark extract as source of antioxidant activity. The strategy of this preliminary work revolved around evaluating the detergency action attributable to a blend of routinely surfactants such as AOS and SCI, and in vitro assessment of antioxidant potential attributable to methanolic extract of cinnamon bark. Further, both of these surfactants that is AOS (Blagojevic, 2016) and SCI (Baumann, 2009; Burnett et al., 2017) have already shown to be environmental friendly with no adverse reactions observed on the surface of soil and water (Baumann, 2009; Blagojevic, 2016; Burnett et al., 2017). Both AOS and SCI are commonly used surfactants in cosmetic preparations (Hibbs, 2007). Additionally, SCI is known to be a gentler surfactant action onto the skin and scalp as these aren’t capable of penetrating onto the skin layer due to larger micellar structure formations (Ghosh & Blankschtein, 2008; Madhaven & Andersen, 2000). Further, mannitol and microcrystalline cellulose were incorporated as anticaking agents that were also reported to be relatively nonirritant and nontoxic in nature (Rouaz et al., 2021; Weiner & Kotkoskie, 1999). The formulation is designed to be used as a reconstitutable powder that needs to be suspended in water prior to application by the user. Since higher viscosity is a desirable attribute, we also incorporated sodium carboxymethyl cellulose as rheology modifier. Data on in vitro detergency action (Figure 2B) the blend of AOS and SCI along with other additives exhibited excellent detergency action. We evaluated whether maltodextrin per se exerts antioxidant effect by assessing its TAC. We observed that the sample of maltodextrin used as filler in the present study possessed the TAC of 1.9 ± 0.16 mg ascorbic acid equivalents/gram (data not shown). Consider that maltodextrin was added at 8.7% in the formulation our data suggest that approximately 0.17 mg ascorbic acid equivalents/gram of the TAC of the formulation containing cinnamon extract is probably attributable to maltodextrin.

Oxidative stress may exert antagonistic effect on hair physiology. Topical treatment of mice with organic peroxide was reported to be associated with early precipitation of the catagen phase of the hair cycle. Further, the study also reported that organic peroxide causes apoptosis in hair follicle cells as well as human epidermal keratinocytes (Hibbs, 2007). Moreover, a clinical trial revealed that application of functional ingredients containing antioxidants was associated with reduced hair shedding and increased total hair count (Davis et al., 2021). We have provided a brief account of studies reporting on association between oxidative stress and hair and scalp homeostasis in the introduction section of the manuscript. Thus, it may be argued that incorporation of antioxidant attributes may be of benefit for hair care. A patent document reports the application of antioxidant shampoo significantly reduced the levels of oxidative stress marker (hydroxyoctadecenedioic acids) in the scalps (Schwartz et al., 2016). Thus, a shampoo with antioxidant attributes may exert beneficial effects on hair health.

In order to confer the formulation antioxidant capabilities, a methanolic extract of Cinnamomum zeylanicum bark was incorporated into the dry powder formulation at 1.2%. Cinnamomum zeylanicum has a long history of utility as a traditional medicine (Ranasinghe et al., 2013). Antioxidant attributes of cinnamon have been well documented in studies of various designs (Mancini-Filho, Van-Koiij, Mancini, Cozzolino, & Torres, 1998; Mathew & Abraham, 2006; Roussel, Hininger, Benaraba, Ziegenfuss, & Anderson, 2009; Sandamali, Hewawasam, Jayatilaka, & Mudduwa, 2021). Cinnamomum zeylanicum is known to contain cinnamaldehyde, α-copaene, α-muurolene, p-methoxycinnamaldehyde and δ cadinene (Abelan et al., 2022). Apart from these, they are also known to be enriched with polyphenols such as catechin, epicatechin, cafeic acids, procyanidin B12, vanillic acid and ferulic acids (Khuwijitjaru et al., 2012). All these primary molecules are known to have antibacterial, antifungal and antioxidant potential (Abelan et al., 2022; Ranasinghe et al., 2013). Recent studies also suggest the use of these extracts to remove dandruff and promote hair growth with improved hair length and density in alopecia patients (Abelan et al., 2022; Mehta, Mehta, & Prasad, 2019). We observed that methanolic extract of cinnamon exhibited appreciable antioxidant activity in vitro (Table 2) and incorporation of the extract into powder shampoo formulation significantly enhanced the antioxidant potential of the powder shampoo (Figures 1A, 1B and 2A). We evaluated total phenolic content of the extract and the formulation containing cinnamon extract as phenolic compounds are known for their antioxidant potential (Miguel-Chávez & Miguel-Chávez, 2017). US10925823B2 describes synergistic antioxidant formulations including that of shampoo (Schwartz, Laughlin, Li, Chen, & Kerr, 2019). US20160346184A1 reveals that levels of hydroxyoctadecenedioic acids are higher in unhealthy scalps and shampoo formulations that decrease hydroxyoctadecenedioic acids in scalp (Schwartz et al., 2016). A kit of shampoo and lotion with anti-inflammatory and antioxidant attributes containing polyphenols from green tea, nanoencapsulated biotechnological blend obtained from Tahiti blue microalgae and Baicapil, flavonoid-rich preparation was reported to increase total follicular unit, number hair and Median hairs per follicular unit (Anzai, Pereira, Malaquias, Guerra, & Mercuri, 2020). However, the above listed formulations do not contain cinnamon extract as source of antioxidant activity. US20220168197A1 describes a powder formulation whose list of ingredients shows Cinnamomum zeylanicum as one of many herbal components used. However, patent doesn’t describe method of processing Cinnamomum zeylanicum. Further, patent doesn’t provide data on antioxidant attributes of cinnamon (Battermann & Kerl, 2021). Given traditional use of cinnamon as a spice and its well-known antioxidant attributes, we opine that it is worth exploring the clinical efficacy of the formulation described herein. Several investigators have attempted at developing hair care formulations that are based on antioxidant attributes or aiming at achieving antioxidant outcomes.

Our study reveals a dry powder shampoo formulation with significant antioxidant activity as a result of incorporation of cinnamon bark extract. The antioxidant shampoo formulation with detergents (AOS and SCI) and anticaking agents described in the present study is appropriate for storage in environmental-friendly packing materials and hence may offer a solution to mitigate burden of use of plastic as storage material for hair cleansing products. The formulation is to be suspended in water just before the use. We opine that the formulation can be explored for antioxidant efficacy in preclinical and clinical settings.

The authors thank Jain (Deemed-to-be University) for support to this study.