Sustainable Use of Extracts of Some Plants Growing in Ethiopia for the Formulation of Herbal Shampoo and Its Antimicrobial Evaluation

: Shampoo prepares surfactants to remove dirt, surface grease, and skin debris from hair, shaft, and scalp without harming the user. This study aimed to formulate herbal shampoo using Ethiopian plant extracts and evaluate their quality parameters. Herbal shampoos were formulated using seed oil extracts of Lepidium sativum L. and Ricinus communis L., and essential oil extracts of Lippia adeonsis Hochst ex. Walp koseret, along with other ingredients. The formulated herbal shampoos were evaluated for their organoleptic and physicochemical properties such as pH, surface tension, viscosity, dirt dispersion, antimicrobial activities, and stability. When the investigation data were assessed, the formulations were pleasant and attractive, produced sufﬁcient foam, and showed suitable cleansing activities. The pH values were in a range 5.90 ± 0.22–7.45 ± 0.19, and lower surface tension (23.39 ± 0.20–31.89 ± 1.04 dyne/cm) together with acceptable viscosity with good shearing properties were obtained. The formulated products exhibited good antimicrobial activities against Escherichia coli, Staphylococcus aureus , and Aspergillus niger. When the surface morphologies of hair samples were examined using a scanning electron microscope (SEM), a clean and smooth surface was observed for treated samples, comparable to that of the marketed shampoo. This result indicated that the formulated herbal shampoo is good, having acceptable properties at par with commercially available shampoos. However, further investigation, research, and development are required to improve its quality and safety.

Another herbaceous plant, Lippa adoensis Hochet. Ex. Koseret (Amharic: Koseret), belongs to the Verbenaceae family and is endemic to Ethiopia [24]. Traditionally, the dried leaves of L. adoensis were used in Ethiopian society as a constituent in the preparation of spiced butter, preservation of cow butter, and food flavoring. In Ethiopian traditional medicine, the leaves of L. adoensis are used to treat several skin disorders such as eczema and superficial fungal infections [25]. The work of Gemeda et al. [26] revealed that the essential oil obtained from the leaves of the plant by hydrodistillation possessed a strong inhibitory effect on the growth of Staphylococcus aureus and Enterococcus hirae, and a moderate effect was observed for Candida albicans and Saccharomyces cerevisiae. Therefore, L. adoensis essential oil extracts can be used in hair-care products such as shampoos, mainly as a preservative, and as a treatment for superficial fungal infections associated with hair and scalp.
Since human hair and scalp are widely exposed to different conditions such as sunlight, environmental pollution, and styling products, it needs regular washing and conditioning to keep hair and scalp feeling and looking clean. Therefore, appropriate shampooing is necessary to overcome such difficulties. However, shampoos most commonly available in markets are synthetic and contain many harsh chemicals associated with different health problems when used repeatedly and continuously [27][28][29]. Therefore, this study aimed to formulate herbal shampoo using extracts of some medicinal plants growing in Ethiopia, and characterize the formulated products for their organoleptic and physicochemical parameters.

Sample Collection and Preparations
The leaves of Lippia adoensis Hochst ex. Walp koseret (hereafter abbreviated as LAK for essential oil extraction) were collected from G/Shasho Kebele, Tarcha Zuriya Woreda, Dawro Zone, South Nation Nationality People Regional State, Ethiopia. The collected leaves were properly packed using a high-density polyethylene bag with a zip lock. The mature dry seeds of R. communis (castor seed) and L. sativum (garden cress) were collected from Merkato market, Addis Ababa, Ethiopia, for seed oil extractions. The three plant materials were identified by a taxonomist at the Department of Biology and Biodiversity Management, College of Natural Sciences of Addis Ababa University, Ethiopia. Other oils, such as eucalyptus oil, coconut oil, and shea butter for shampoo formulations, were bought from Ethiopian Skin Beauty Secret, Addis Ababa, Ethiopia.

Chemicals, Reagents, and Apparatus
All chemicals used in the present work were of analytical grade, and all glassware was used after thorough washing and drying in an oven. The chemicals used included anhydrous sodium sulfate (Na 2  First, freshly collected LAK leaves were thoroughly washed with tap water followed by distilled water, shed-dried, and crushed into powder using an electrical grinder. About 150 g of powder was taken into a 2 L clean, dry round bottom flask. Hydrodistillation was carried out after adding 1.5 L water and boiling chips for 3 h to complete the essential oil extraction using a commercial Clevenger-type apparatus. The extraction was conducted several times to collect enough oil for the characterization and formulation of the product. The oil sample obtained from each hydrodistillation was freed from moisture by adding enough anhydrous sodium sulfate. The resulting moisture-free essential oil sample was stored in the refrigerator at 4 • C for further analysis. The percent yield of the extracted essential oil from each sample based on various LAK leaf samples was determined using the following formula [30][31][32]:

Extraction of Seed Oil
Mature, dry seeds of L. sativum and R. communis were ground into powder using an electric grinder. The seed oil extraction was conducted using an automatic Soxhlet extraction unit (Extraction Unit E-816 ECE, BUCHI Laboratories AG, Flawil, Switzerland). Twenty grams of the powdered seeds were placed into six cellulose thimbles and then transferred into each extraction chamber. About 150 mL of PET ether (40-60 • C) was added to each clean, dry extraction beaker containing boiling chips, and the extraction was conducted by setting the extraction time to 3 h, rinsing time to 5 min, and drying time to 20 min. At the end of the extraction, the solvent sent into the solvent tank using the instrument during the drying step was collected to a clean, dry flask and subsequently used for further extraction. Finally, the seed oils from each extraction beaker, containing a small amount of extraction solvent, were combined and further concentrated using a rotary evaporator. The amount of oil recovered and its percentage in the original sample were calculated as follows [18,33]: Oil content (%) = Mass of oil extracted (g) Sample weight (g) × 100 (2)

GC-MS Analysis of LAK EO
The chemical composition analysis of LAK EO was conducted using gas chromatography (Thermo) hyphenated with a mass detector (GC-MS), at Shivaji University, Kolhapur, India. The GC apparatus equipped with a capillary column (length = 30 m, i.d. = 0.32 mm, and film thickness of 0.25 µm), coupled to a mass spectrometer (MS) type DSQII (Thermo) with a 70 eV detector impact of electrons was used. The carrier gas was helium at a rate of 1 mL min −1 ; the injection volume was 1 µL; injector split mode 1:70. The injector Sustainability 2023, 15, 3189 5 of 21 temperature was 270 • C and the GC temperature program was held at 40 • C for 1 min, raised to 270 • C at a rate of 3 • C/min, and then held at 270 • C for 2 min. The mass spectra of each compound were scanned across the m/z range of 50 to 650. Identification of the compounds was obtained by comparing their mass spectra and retention times with those of standards (NIST14.lib) and those found in literature [34].

Formulation of Herbal Shampoos
Four different herbal shampoo formulations were prepared according to the proportion given in Table 1. The shampoos were formulated by applying the methods specified by Patel et al. [5] and Sawarkar et al. [35] with slight modifications. Beeswax, coconut oil, and shea butter were allowed to melt in a hot water bath at 75 • C. The oils were mixed with castor oil and L. sativum in a 250 mL clean, dry round bottom flask and saponified with KOH solution using a reflux condenser by heating the flask at 75 • C for about 30 min. The weighed amounts of SLS were added to the mixture with continuous stirring using a mechanical stirrer. Thickeners and foam stabilizers (acacia gum, CMC, EDTA), stirred in the water phase for 30 min, were added to the above solution with continuous stirring. Finally, the LAK and eucalyptus essential oil were added to the shampoo solution after lowering the temperature to 35-40 • C, mixed thoroughly, allowed to cool to room temperature, and transferred to clean, dry shampoo bottles for further evaluation and characterization. F-1 = herbal shampoo formulation-1; F-2 = herbal shampoo formulation-2; F-3 = herbal shampoo formulation-3; F-4 = herbal shampoo formulation-4; and q.s. = quantum satis.

Evaluation of Herbal Shampoos
Different quality control tests, including organoleptic and physicochemical characteristics such as visual tests, pH, solid contents, and viscosity tests, were conducted for the formulated herbal shampoos to assess the quality of the product. In addition, specific tests for the formulations, including surface tension, foam volume, foam stability, dirt dispersion, surface morphology, skin sensitization tests, and preliminary stability study, were also carried out.

•
Physical Appearance/Visual Inspection: Physical appearance tests (organoleptic evaluation) on parameters, e.g., color, clarity, and odor, were carried out. These parameters were evaluated visually and via the smelling sensation, respectively. • pH Determination: The pH of shampoo (10% shampoo solutions) was determined in distilled water at room temperature using a calibrated pH meter [27,36]. Triplicate measurements were taken for each formulation to report mean ± standard deviation. • Dirt Dispersion Test: Two drops of shampoo were added to a large test tube containing 10 mL of distilled water. A dirt dispersion test was performed by adding a drop of India ink and then shaking the content. The amount of ink concentrated in the foam portion after shaking ten-times was then estimated as none, light, or heavy [37,38]. • Surface Tension Measurement: It has been stated that a good shampoo should reduce the surface tension of pure water to about 40 dynes/cm [39]. Surface tension lowering is one of the mechanisms concerned with detergency. The surface tension measurement of shampoo was conducted by the drop-count method using a stalagmometer. The latter was first rinsed thoroughly using a solution of chromic acid in distilled water and then with distilled water alone, as surface tension is highly affected by grease or other lubricants. The measurements were carried out with a 10% shampoo dilution in distilled water at room temperature. First, distilled water was sucked into the clean, dry stalagmometer until it reached the upper mark, and then the number of drops was recorded by counting until the liquid passed from the upper to the lower mark. Similar procedures were followed for 10% shampoo solutions, and the data for surface tension were calculated by applying Equation (3) [39]. The measurements were repeated thrice and reported as mean ± standard deviation.
where W 1 is the weight of the empty beaker (g), W 2 is the weight of the beaker with distilled water (g), W 3 is the weight of the beaker with shampoo solution (g), n 1 is the number of drops of distilled water, n 2 is the number of drops of shampoo solution, R 1 is the surface tension of distilled water at room temperature = 72.8 dynes/cm, and R 2 is the surface tension of shampoo solution (dynes/cm). • Viscosity Measurement: About 100 mL of prepared shampoo solutions were taken in 250 mL of clean, dry beakers. The solution was then placed on a viscometer stand and allowed to stir with spindle #3. The viscosity of formulations was measured at varying revolutions per minute (rpm) to test the pseudo-plasticity of the preparation using an NDJ rotational viscometer. The viscosity of each formulation was recorded at varying speeds (i.e., 0.3, 0.6, 1.5, 3, 6, and 12 rpm) at room temperature (23.5 • C). The study kept the temperature and container size constant [40]. • Determination of Percentage Solid Contents: The exact weight of the shampoo was determined. The weight of a clean, dry evaporating dish with an evaporating 4 g shampoo dish was recorded. The evaporating dish with shampoo was then placed on the hot plate until complete evaporation of the liquid portion [41,42]. The weight of the remainder after drying was calculated, and the percent solid contents of shampoo were determined as per the following formula: • where M 1 is the weight of the wet sample (before drying (g)) and M 2 is the weight of the dry sample (after drying (g)).

•
Foaming and Foam Stability: The cylinder shake method was used to study shampoo formulation foaming capacities and foam stabilities [43][44][45]. About 50 mL of 1% formulated shampoo solution were taken in a 250 mL capacity graduated cylinder, and the content was shaken vigorously ten times. The foam stability was measured by recording the foam volume produced by the shake test after 1, 3, and 5 min, respectively. The amount of foam each shampoo produced was recorded. • Skin Irritancy: A skin irritancy test was conducted by applying 1% v/v shampoo solution in distilled water over the skin. The applied shampoo solution remained in contact for about 2 h, and a response of any redness, itching, and inflammation was observed [27]. modifications. The shampoo samples were put in a humidity chamber at a temperature of 40 ± 2 • C and a relative humidity of 75% for two months [2,46]. The same samples were stored at room temperature (25 ± 2 • C) for two months. All samples were inspected for their organoleptic properties and physicochemical parameters such as pH, surface tension, viscosity, and foaming at the end of the storage period [42,47]. • Analysis of Dirt Cleaning Effectivity of Herbal Shampoo by SEM: The surface morphology of hair samples before and after treatment with formulated and marketed shampoos was examined using a scanning electron microscope (SEM). Three hair samples: untreated hair (S-1), hair treated with herbal shampoo formula, F-4 (S-2), and hair treated with marketed shampoo (Head and Shoulders) (S-3), were prepared for SEM analysis as follows. The hair strands were dipped in a 250 mL graduated cylinder containing 20 mL of the shampoo solution, and continuously shacked for about 10 min. The solution was drawn off, and the hair strands were rinsed with 20 mL of distilled water for 1 min. The rinsing was repeated continuously, and the hair samples were dried by slightly pressing between pieces of filter paper [46]. The dried hair samples were cut into small strands and mounted directly on the SEM sample stub by using double-side stitching carbon tape. The photomicrographs at appropriate magnification were obtained for surface characterization.

Antimicrobial Activities
• Bacterial and Fungal Strains: The antibacterial and antifungal activities of three herbal shampoo formulations and one LAK essential oil were carried out using two pure bacterial cultures: Staphylococcus aureus (ATCC-25923) and Escherichia coli (ATCC-25922), and one clinical fungal isolate of Aspergillus niger. Mueller-Hinton agar (MHA) medium and potato dextrose agar (PDA) were used to conduct the screening for antibacterial and antifungal activity, respectively [25,48]. • Disc Diffusion Assay • Antibacterial Activity Studies: The antifungal and antibacterial assay was conducted using the disc diffusivity method. Fluconazole and gentamycin antibiotics were used as positive control for fungal and bacterial tests, respectively. The antibacterial activities against E. coli and S. aureus were studied using a diffusion disc over an MHA medium. Diffusion discs nearly 6 mm in diameter were prepared from Whatman No. 1 filter paper using a puncher, sterilized by spraying with 70% ethanol, autoclaved, and air-dried inside the hood [49,50]. The pure active bacterial culture of E. coli and S. aureus was inoculated into two separate Petri plates containing MHA media using a sterile loop. Each plate was then properly spread aseptically using a sterile spreader and divided into four quadrants. The previously prepared disc was aseptically placed into each quadrant using sterile forceps. About 10 µL of test solutions and positive control were poured with a sterile micropipette into each disc. The plates were covered adequately with parafilm and kept in an incubator at 37 • C for 24 h. The zone of inhibition (ZOI) was measured after 24 h of incubation using a scale, and the experiment was repeated thrice. • Antifungal Activity Studies: The fungal pure culture of A. niger was obtained from the Food Process Engineering Department, Addis Ababa Science and Technology University. A colony of seven-day-old pure cultures was taken with a sterile loop and dissolved in 5 mL of distilled water. The pure cultures were then inoculated into sterile Petri plates over a PDA medium using a 3 mm sterile cork borer. Then, the plates were properly covered with parafilm and incubated for seven days at 28 • C. The fungal suspension was properly shaken with a vortex shaker and about 100 µL suspensions were poured with a sterile micropipette into Petri plates containing PDA medium. It was spread over the medium using a sterile spreader. The 6 mm sterile discs were placed in Petri plates using sterile forceps, and 50 µL test solutions and fluconazole control were charged into each disc. The plates were covered with parafilm and incubated at 28 • C. The experiment was repeated twice, and ZOI was measured using scales after 48 h incubation.

Statistical Analysis
Statistical analysis of data was conducted using IBM SPSS statistics 20. One-way analysis of variance (ANOVA) was used to determine significance, and p values < 0.05 were considered significant. Triplicate measurements were carried out for all tests, and data were stated as mean ± standard deviation.

Essential and Seed Oil Yields
The percentage oil yields (%w/w) of both essential and seed oils are given in Table 2. The essential oil of LAK obtained by hydrodistillation was yellow, having a pleasant smell with an average yield of 1.17 ± 0.20%. The collected plant leaf species possessed a better yield when compared to the previous report of essential oil of the leaves collected from East Wellega, Ethiopia. The latter was obtained by hydrodistillation with a yield of 0.7 ± 0.116% [26]. The seed oil contents of L. sativum and R. communis were 20.45 ± 2.79% and 27.74 ± 0.88%, respectively. The oil yield of L. sativum in the present study was lower than the previous report of Solomon et al. [51], which was 23.72%. These variations in the yield of essential and seed oils may be related to variations in soil types, weather conditions, and other growth conditions. The oil yield of R. communis obtained in this study was better than that of Gebrehiwot and Zelelew [33]. They reported that the plant seeds collected from farmland around Abiy Adi, central province, 95 km from the capital of Tigray, Ethiopia, had an oil yield of 9.25%. The variation in plant nutrient and water supply during crop growth, climatic conditions during seed development and maturation, and the harvest and drying practices are of considerable significance for the difference in yield. The percent yield of oils (expressed as weight extracted oil per weight of sample charged). Each reading is the average of three determinations ± standard deviations with p < 0.05.

Essential Oil Composition
As shown in Table 3, seven different compounds were identified by gas chromatographymass spectrometry (GC-MS), including six monoterpene hydrocarbons and one ester. The oil contained linalool (73.13%) as the dominating compound, which coincides with work by Workalemahu et al. [52], who reported that the major constituent (72.85%) of the oil was linalool when analyzed by GC-MS. 1,8-cineole (10.42%), 1,6-Octadien-3-ol-3,7-dimethylformate (7.44%) and cyclohexanol, 1-methyl-4-(1-methylethenyl)-, cis-(7.06%) were the other major constituents of the oil. Linalool, a monoterpene alcohol, is a colorless to slightly yellow liquid, and can be found in many essential oils, such as that of lemon, spearmint, rose, cinnamon, and cypress [53]. According to Li et al. [54], linalool is the main constituent of scented herbs and rosewood, and is responsible for the characteristic aroma of citrus fruits. Another work by Herman et al. (2015) revealed that linalool is the major constituent of many essential oils such as T. vulgaris (2.2%), J. communis (10.9%), P. graveolens (10.5%), C. bergamia (8.8%), C. paradise (1.1%), L. angustifolia (30.1-46.3%), and C. zeylanicum (8.5%). About 60-80% of perfumed hygiene products and cleaning agents such as soaps, detergents, shampoos, and lotions contain linalool as a fragrance agent [55]. It has been reported that adding linalool to essential oils may significantly enhance their antimicrobial effectiveness and reduce their concentrations in products; it is a chemical intermediate to yield vitamin E, a hypotensive, perfumed product, and a cleaning agent [54]. It is used as a flavoring agent, insecticide, and fragrance in hair-care products, personal care products, and cleaning and laundry products [53]. Eucalyptol (1,8-cineole) is another important monoterpenoid in LAK EO; it is the active constituent of eucalyptus essential oil [56,57] and predominating components of Amomum subulatum Roxb. [58]. Eucalyptol has been reported for its effectivities in antioxidant, antiinflammatory, antibacterial, antifungal, and antiaflatoxigenic activities [59,60]. The oil also contains a small amount of D-Limonene (0.48%), a common compound in lemon and other citrus fruits. According to Sarkic and Stappen [53], linalool, as well as limonene, can cause allergenic dermatitis when oxidized and was classified as an allergenic fragrance by European Union (EU) directive. Therefore, it should be used at the lowest possible concentration in personal care products such as shampoos that might have the capacity to enhance the antimicrobial, antioxidant, and anti-inflammatory activities of the products. Monoterpenes were also identified from the essential oil of LAK, which has been reported to exhibit antimicrobial, anti-inflammatory, and antioxidant activities, as shown in Figure 2.

Herbal Shampoo Formulations
Liquid herbal shampoo formulations were prepared using extracts and oils from plants commonly grown in Ethiopia. To evaluate their antimicrobial effect and other phys icochemical properties, four different formulations were prepared by varying the concen trations of some ingredients, i.e., LAK, castor oil, and L. sativum seed oil, according to th proportions given in Table 1. The formulations were prepared by first undertaking sapon ification of oils with the calculated amount of KOH and mixing other ingredients with continuous stirring using a mechanical stirrer. All prepared formulations were nontrans parent and yellowish, with good viscosity and pleasant smell (Figure 3).

Herbal Shampoo Formulations
Liquid herbal shampoo formulations were prepared using extracts and oils from plants commonly grown in Ethiopia. To evaluate their antimicrobial effect and other physicochemical properties, four different formulations were prepared by varying the concentrations of some ingredients, i.e., LAK, castor oil, and L. sativum seed oil, according to the proportions given in Table 1. The formulations were prepared by first undertaking saponification of oils with the calculated amount of KOH and mixing other ingredients with continuous stirring using a mechanical stirrer. All prepared formulations were nontransparent and yellowish, with good viscosity and pleasant smell (Figure 3).

Herbal Shampoo Formulations
Liquid herbal shampoo formulations were prepared using extracts and oils from plants commonly grown in Ethiopia. To evaluate their antimicrobial effect and other physicochemical properties, four different formulations were prepared by varying the concentrations of some ingredients, i.e., LAK, castor oil, and L. sativum seed oil, according to the proportions given in Table 1. The formulations were prepared by first undertaking saponification of oils with the calculated amount of KOH and mixing other ingredients with continuous stirring using a mechanical stirrer. All prepared formulations were nontransparent and yellowish, with good viscosity and pleasant smell (Figure 3).

Herbal Shampoo Evaluation
The herbal shampoo formulations were subjected to different characterization parameters to ensure their quality and safety. Results for the evaluated parameters are thoroughly discussed in the following sections.

Organoleptic Properties
The organoleptic properties of shampoo formulations are important as consumers seek a well-formulated product with an elegant nature. They prefer the product with an acceptable fragrance, good clarity, and attractive color. The formulated shampoo was subjected to visual inspection (color, odor, and clarity) ( Table 4). The use of EOs as active

Herbal Shampoo Evaluation
The herbal shampoo formulations were subjected to different characterization parameters to ensure their quality and safety. Results for the evaluated parameters are thoroughly discussed in the following sections.

Organoleptic Properties
The organoleptic properties of shampoo formulations are important as consumers seek a well-formulated product with an elegant nature. They prefer the product with an acceptable fragrance, good clarity, and attractive color. The formulated shampoo was subjected to visual inspection (color, odor, and clarity) ( Table 4). The use of EOs as active ingredients in cosmetics like shampoo helps to impart perfumery (fragrances) and aromatherapy to the products [61]. Accordingly, all the formulations possessed a good fragrance due to the presence of eucalyptus essential oil as a scent. The LAK essential oil itself imparts a pleasant aroma to the shampoos and has been traditionally used as a food flavoring agent and preservative [62]. Among the four formulations, herbal shampoo F-4 possessed the strongest LAK essential oil aroma and contained the highest concentration (i.e., 12%) compared to the first three formulations. No synthetic scenting agents or colorants were used in the shampoos, being more or less associated with different side effects. Each reading is the average of three determinations ± standard deviations with p < 0.05.

pH and Surface Tension
Since it helps to identify whether the chemical substances are acidic, basic, or neutral, pH is an important parameter. These properties are solely linked to the efficacy and safety of the products, e.g., shampoos. The pH of shampoo plays a pivotal role in manipulating hair quality, reducing the irritation of the mucous membrane, and enhancing the biological functioning of the scalp [37,45]. The normal pH of the scalp is 5.5 [28]; however, most reports revealed that the pH of shampoo formulations within the range 5.0-7.8 was ideal, being neutral and good to use on hair and scalp [6,63]. pH of the formulated herbal shampoos was in the range 5.90-7.45 (Table 4), which coincides with literature reports for ideal shampoo formulations (i.e., 5.0-7.8). The pH of shampoo formulations can be balanced using citric acids, as mild acidity prevents swelling and endorses constriction of the scales, thereby bringing shininess [36,37]. However, more acidic solutions can cause the cuticle shrinking (the outer layer of hair), and lay flattered on the hair shaft [36]. On the other hand, basic shampoo solutions can cause cuticle swelling and opening, thus reducing the performance of the product in terms of compatibility, irritation, friction, and frizz effect [64].
The surface tension of the shampoo formulations was measured by the drop-count method using a stalagmometer. It has been reported that the ideal shampoo solution should reduce the surface tension of pure water from 72.8 dyne/cm to about 40 dyne/cm [2,37]. Since water molecules at the surface do not have other water molecules above the surface, it experiences a greater pulling force from water molecules below the surface rather than above. The tension between these pulling forces (surface tension) is the highest for water molecules due to hydrogen bonding. Since the detergent molecule in shampoo solution possesses two ends (hydrophobic and hydrophilic), the hydrophobic end pushes away from the water surface. It weakens hydrogen bonds holding the water molecules at the surface and results in the breaking of water surface tension. Therefore, shampoo solutions clean greases by lowering the surface tension of water. According to Table 4, the surface tension of herbal shampoo formulations ranged from 23.39 to 31.89 dyne/cm, which is consistent with the research work of Shreya and Kalpana [29]. This is a reasonable implication that the formulated herbal shampoo preparations possessed good detergency.

Dirt Dispersion Test and Percent Solid Contents
A dirt dispersion test was conducted by adding a drop of India ink to the test tubes containing a shampoo solution. The amount of ink concentrated in the foam region was observed. All formulated shampoos succeeded in concentrating ink in the water part (Figure 4), implying that the products possessed sufficient cleansing activities. However, herbal shampoo formulations (F-1 and F-4) showed relatively better cleansing activities according to the dirt dispersion test, as they produced transparent foam in the presence of ink (considered as dirt). The better dirt dispersion effectivity of the herbal shampoo F-1, shown in Figure 4, might be related to the high concentration of surfactant (i.e., SLS) added during the formulation when compared to the other formulations. This returns to the fact that dirt removal activities of shampoo formulations are directly related to the concentration of surfactants used.

Dirt Dispersion Test and Percent Solid Contents
A dirt dispersion test was conducted by adding a drop of India ink to the test tubes containing a shampoo solution. The amount of ink concentrated in the foam region was observed. All formulated shampoos succeeded in concentrating ink in the water part (Figure 4), implying that the products possessed sufficient cleansing activities. However, herbal shampoo formulations (F-1 and F-4) showed relatively better cleansing activities according to the dirt dispersion test, as they produced transparent foam in the presence of ink (considered as dirt). The better dirt dispersion effectivity of the herbal shampoo F-1, shown in Figure 4, might be related to the high concentration of surfactant (i.e., SLS) added during the formulation when compared to the other formulations. This returns to the fact that dirt removal activities of shampoo formulations are directly related to the concentration of surfactants used. The dirt dispersion test is a crucial parameter for the evaluation of the cleansing activities of shampoos. Since ink or dirt that stays in foam is difficult to rinse away and will redeposit on the hair, the shampoo solutions that cause the ink to stay in the foam region are considered lower quality [2,65]. Thus, for good quality shampoos, the dirt or ink The dirt dispersion test is a crucial parameter for the evaluation of the cleansing activities of shampoos. Since ink or dirt that stays in foam is difficult to rinse away and will redeposit on the hair, the shampoo solutions that cause the ink to stay in the foam region are considered lower quality [2,65]. Thus, for good quality shampoos, the dirt or ink should stay in the water part, so that cleansing becomes easy.
The percent solid contents of shampoo solutions were determined by evaporating all liquid portions using hot plates. The percentage of solid contents was in the range 26.15-32.36%, as shown in Table 4. Accordingly, the result revealed that the formulations contained optimum solids compared to the literature values (i.e., 20.0-30.0%) [6,66]. Herbal shampoo (F-1) possessed lower solid content (26.15%) than F-2 (32.36%), followed by F-3 (29.87%) and F-4 (28.93%). A well-formulated shampoo may contain percent solid contents of 20.0-30.0% as lower amounts would result in a watery formulation, which can be washed away quickly, whereas shampoos with high solids amounts would be difficult to wash out [6,67].

Viscosity
To detect the pseudo-plastic nature, the viscosity of formulated herbal shampoos was measured at varying speeds (0.3-12.0 rpm) [40,68], as shown in Table 5. As seen in Figure 5, the rheological evaluation results indicated that the formulations' viscosity changed gradually when the speed (rpm) varied. This means that the shampoo formulations were shear thinning or pseudo-plastic because the viscosity decreased with an increase in rpm. The pseudo-plastic nature is a desirable attribute in shampoo formulation, which is directly related to the spreadability of shampoo on hair [37]. All formulated herbal shampoos followed a pseudo-plastic rheogram. However, the viscometer did not show any reading for F-2 and F-3 at 0.3 rpm, as seen in Figure 5. This might be due to the high viscosity of these two formulations, which make them resist the spindle rotation at a very low rotational speed (i.e., 0.3 rpm). The viscosities of formulated herbal shampoos at 12 rpm were at acceptable ranges compared to the findings in the work by Shreya and Kalpana [29]. These authors reported that the viscosities of polyherbal shampoos measured were in the range 1260-9048 mPa·s. Viscosity determines the thickness or stickiness, and the resistance of the liquid to flow. Viscosity measurement is an important parameter as it plays a crucial role in defining and controlling many product attributes, such as shelf-life stability, clarity, ease of flow, consistency, and spreadability upon application on hair [6,67]. Therefore, shampoo formulations with controlled viscosities are important in defining product homogeneity and stability. (-) = no viscosity values at lower rpm; F-1 = herbal shampoo formula-1; F-2 = herbal shampoo formula-2; F-3 = herbal shampoo formula-3; and F-4 = herbal shampoo formula-4. Viscosity determines the thickness or stickiness, and the resistance of the liquid t flow. Viscosity measurement is an important parameter as it plays a crucial role in defin ing and controlling many product attributes, such as shelf-life stability, clarity, ease o flow, consistency, and spreadability upon application on hair [6,67]. Therefore, shampo formulations with controlled viscosities are important in defining product homogeneit and stability.

Foaming and Foam Stability
The result of foaming capacities and foam stabilities of the formulated herbal sham poos are indicated in Table 6. All formulations produced sufficient foam, and foam stabil ity was determined by reading the foam volume on a 1 min interval for 5 min. As see from Table 6 and Figure 6, the foam produced decreased within the first three minute and remained constant afterward. The foam stability of formulations might be due to th foam stabilizing agent (i.e., acacia gum powder) used in the formulations. Shampoo for mulations with sufficient foam are important in commercialization since customers prefe good foaming shampoos even though foam generation has less relation with the cleansin activities [69]. Table 6. Foam volume and foam stability.

Foaming and Foam Stability
The result of foaming capacities and foam stabilities of the formulated herbal shampoos are indicated in Table 6. All formulations produced sufficient foam, and foam stability was determined by reading the foam volume on a 1 min interval for 5 min. As seen from Table 6 and Figure 6, the foam produced decreased within the first three minutes and remained constant afterward. The foam stability of formulations might be due to the foam stabilizing agent (i.e., acacia gum powder) used in the formulations. Shampoo formulations with sufficient foam are important in commercialization since customers prefer good foaming shampoos even though foam generation has less relation with the cleansing activities [69]. Table 6. Foam volume and foam stability.  F-1  140  130  125  120  120  120  F-2  120  120  115  115  110  110  F-3  100  90  90  90  85  85  F-4  60  50  40  40  40  40 F-1 = herbal shampoo formula-1; F-2 = herbal shampoo formula-2; F-3 = herbal shampoo formula-3; and F-4 = herbal shampoo formula-4.
Sodium lauryl sulfate (SLS), used as a surfactant in shampoo formulations, also participates in foaming. The foam formation (volume) decreased when changing from F-1 to F-4 ( Figure 6). This may be due to the decreasing concentration of SLS added while formulating the shampoos.
3; and F-4 = herbal shampoo formula-4. Sodium lauryl sulfate (SLS), used as a surfactant in shampoo formulations, also p ticipates in foaming. The foam formation (volume) decreased when changing from F-F-4 ( Figure 6). This may be due to the decreasing concentration of SLS added while mulating the shampoos.

Skin Irritation Test
A good quality shampoo is considered to be user-friendly and does not cause ha ful side effects. It should not irritate the mucous membrane, such as the eye or nose, a should not cause redness, itching, dryness, or shedding of skin. The skin irritancy test w conducted by directly applying the product to the skin. The result revealed no sign itching, redness, or inflammation for tested shampoo formulations (F-1 and F-4). The fore, the tested herbal shampoo formulations were safe to be used. However, furthe vivo and in vitro irritancy studies on eyes and other mucous membranes were requi to ensure complete compliance with the products.

SEM Analysis of Dirt Cleaning Effectivity of Herbal Shampoo
The surface morphology of the hair samples before and after treatment with form lated herbal shampoo and marketed "Head and Shoulders" shampoo was studied us SEM. The result of the cleansing activity study of shampoo solution examined via SEM shown in Figure 7. The photograph of the untreated hair sample was compared to tha hair treated with one herbal shampoo formulation and one marketed shampoo. The sults from SEM revealed that the untreated hair sample (Figure 7a) had a scrambled pearance due to the desquamation of the hair cuticle and the existence of dirt on its surf [66,70].
Both formulated and marketed shampoos exhibited good cleansing properties, as served from microphotographs (Figure 7b,c), which agree with previous work [66]. The fore, treating hair with shampoos containing natural ingredients is helpful in cleansi conditioning, protecting the hair's morphological integrity, and providing consiste and shine to the hair. SEM microphotography of hair is important for studying

Skin Irritation Test
A good quality shampoo is considered to be user-friendly and does not cause harmful side effects. It should not irritate the mucous membrane, such as the eye or nose, and should not cause redness, itching, dryness, or shedding of skin. The skin irritancy test was conducted by directly applying the product to the skin. The result revealed no sign of itching, redness, or inflammation for tested shampoo formulations (F-1 and F-4). Therefore, the tested herbal shampoo formulations were safe to be used. However, further in vivo and in vitro irritancy studies on eyes and other mucous membranes were required to ensure complete compliance with the products.

SEM Analysis of Dirt Cleaning Effectivity of Herbal Shampoo
The surface morphology of the hair samples before and after treatment with formulated herbal shampoo and marketed "Head and Shoulders" shampoo was studied using SEM. The result of the cleansing activity study of shampoo solution examined via SEM is shown in Figure 7. The photograph of the untreated hair sample was compared to that of hair treated with one herbal shampoo formulation and one marketed shampoo. The results from SEM revealed that the untreated hair sample (Figure 7a) had a scrambled appearance due to the desquamation of the hair cuticle and the existence of dirt on its surface [66,70].
Both formulated and marketed shampoos exhibited good cleansing properties, as observed from microphotographs (Figure 7b,c), which agree with previous work [66]. Therefore, treating hair with shampoos containing natural ingredients is helpful in cleansing, conditioning, protecting the hair's morphological integrity, and providing consistency and shine to the hair. SEM microphotography of hair is important for studying the cleansing activities of shampoos [70], as it clearly shows the appearance of the hair surface before and after treatment with shampoo solutions.

Stability Study
Stability is an important parameter to ensure the quality and safety of products during storage times. An accelerated stability study was conducted with required modifications according to ICH guidelines. In accelerated stability studies, the products are subjected to different stress conditions, such as temperature (higher than ambient), moisture, pH, light, agitation, etc., which accelerate product degradation [71]. Accelerated stability testing (at relatively high temperatures and/or humidity) is used to determine the type of degradation products that may be found after long-term storage. The formulated products were stored at room temperature and in a humidity chamber at 40 ± 2 • C and 75% relative humidity for two months. During this storage period, the products were assessed for their organoleptic and physicochemical parameters at the end of each second week. Two formulated herbal shampoos (F-1 and F-4) were tested for different parameters, i.e., color, odor, pH, viscosity, and surface tension, and the results are reported in Tables 7 and 8. There was no significant change in tested parameters when compared to those stored at room temperature. As a result, the stability and acceptability of organoleptic and physicochemical properties of formulations during storage indicated the physical and chemical stability of developed formulations. cleansing activities of shampoos [70], as it clearly shows the appearance of the hair surface before and after treatment with shampoo solutions.

Stability Study
Stability is an important parameter to ensure the quality and safety of products during storage times. An accelerated stability study was conducted with required modifications according to ICH guidelines. In accelerated stability studies, the products are subjected to different stress conditions, such as temperature (higher than ambient), moisture, pH, light, agitation, etc., which accelerate product degradation [71]. Accelerated stability testing (at relatively high temperatures and/or humidity) is used to determine the type of degradation products that may be found after long-term storage. The formulated products were stored at room temperature and in a humidity chamber at 40 ± 2 °C and 75% relative humidity for two months. During this storage period, the products were assessed for their organoleptic and physicochemical parameters at the end of each second week. Two formulated herbal shampoos (F-1 and F-4) were tested for different parameters, i.e., color, odor, pH, viscosity, and surface tension, and the results are reported in Tables 7 and 8. There was no significant change in tested parameters when compared to those stored at   Surface tension and pH readings are the averages of three measurements ± standard deviations with p < 0.05, and viscosity measurement was obtained @12 revolutions per minute using a rotational viscometer with spindle #3.

Antimicrobial Activities
Antimicrobial activities of the formulated herbal shampoos and LAK essential oils were studied against two pathogenic bacterial isolates (E. coli and S. aureus) and fungal isolates of A. niger. It was recently reported that antimicrobial materials such as silver nanoparticles (AgNPs) [72], different plant extracts [73], and synthesized polymeric materials [74] are developed to be used as antimicrobial agents for healing various disorders caused by microbes.
The tests were carried out using disc diffusion assay over MHA and PDA medium for bacteria and fungus, respectively. Each shampoo formulation (F-1, F-4, and F-4 with silver nanoparticles, AgNPs) and LAK essential oil were used in the concentrated form [46]. Herbal shampoo F-4 added to AgNPs (5 mL shampoo in 1 mL AgNPs (1 mg/mL)) was used in addition to formulas F-1 and F-4 to compare the effect of nanoparticles on the antimicrobial activities of shampoo [75]. Both antifungal and antibacterial activity studies were conducted by the disc diffusivity method using a 6 mm Whatman No. 1 filter paper disc [66]. The results of the measured inhibition zone of both test organisms are indicated in Table 9. Herbal shampoos formulated using different herbs, and LAK EO possessed good antibacterial activities against S. aureus (Figure 8) (ZOI = 8.00 ± 0.00-14.33 ± 0.58) and antifungal activities against A. niger (ZOI = 28.33 ± 1.15-30.00 ± 2.00) ( Table 9). The Gramnegative bacteria (E. coli) tested were resistant to all shampoo formulations. The effectivities of formulated herbal shampoos against S. aureus and A. niger are mainly associated with the compositions of shampoo formulations, as they were formulated using different herbs such as castor oil, beeswax, and coconut oil in addition to LAK EO that already possesses antibacterial and antifungal activities. As seen from stituent of many essential oils, possessed high antimicrobial effectivities against Candida albicans, Escherichia coli, and Staphylococcus aureus. In addition, Guleria et al. [79] isolated linalool and other monoterpenes (having good antimicrobial activities) from essential oil extracts of Zanthoxylum alatum using repeated preparative thin-layer chromatography (TLC) and identified by GC-MS analysis. Eucalyptol (1,8-cineole), another dominating compound identified by GC-MS, has been reported to possess good antimicrobial activities [57,60]. Therefore, high sensitivities of tested fungus and bacteria to essential oil might be related to the presence of eucalyptol and other terpenoids, in addition to phenolic, aliphatic, and aromatic hydrocarbons. One interesting biological effect of LAK EO oil is its antimicrobial activities related to its chemical compositions. Among tested microorganisms, E. coli was the most sensitive to LAK EOs, while being more resistant to all tested shampoos (Figure 9). E. coli, one category of Gram-negative bacteria, showed a high sensitivity to LAK EOs even though the report of Nazzaro et al. [76] described that more EOs are powerful against Gram-positive than Gram-negative bacteria. The major constituent of LAK EO analyzed by GC-MS was linalool (73.13%), 1,8-cineole (10.42%), and linalyl formate (7.44%), as seen in Table 6. In this regard, Peana et al. [77] and Herman et al. [78] revealed that linalool, the major constituent of many essential oils, possessed high antimicrobial effectivities against Candida albicans, Escherichia coli, and Staphylococcus aureus. In addition, Guleria et al. [79] isolated linalool and other monoterpenes (having good antimicrobial activities) from essential oil extracts of Zanthoxylum alatum using repeated preparative thin-layer chromatography (TLC) and identified by GC-MS analysis. Eucalyptol (1,8-cineole), another dominating compound identified by GC-MS, has been reported to possess good antimicrobial activities [57,60]. Therefore, high sensitivities of tested fungus and bacteria to essential oil might be related to the presence of eucalyptol and other terpenoids, in addition to phenolic, aliphatic, and aromatic hydrocarbons.  The resistance of microorganisms to conventional chemicals and drugs is in an esca lating increase. Thus, new biocides with wide activities and safety inspired many re searchers and are currently becoming their great focus. Essential oils from various plan The resistance of microorganisms to conventional chemicals and drugs is in an escalating increase. Thus, new biocides with wide activities and safety inspired many researchers and are currently becoming their great focus. Essential oils from various plant parts, such as leaf, root, stem, bark, and flower, possess broad classes of organic compounds. They are secondary plant metabolites composed of variable amounts of terpenes, terpenoids, alcohols, aldehydes, phenolic acids, and aliphatic hydrocarbons [80][81][82]. Essential oils having these metabolites have good capacities in inhibiting or slowing bacteria growth, yeasts, and molds. This increases opportunities to use them as a food preservative and in pharmaceutical products [76,[81][82][83].

Conclusions
The shampoo segment undoubtedly comprises the largest unit sale among hair-care products as it participates in cleansing and beautification. Indeed, shampoo comprises a variety of ingredients that participate in its formulation. However, synthetic preservatives, antimicrobial agents, and conditioners have sometimes been the cause of adverse effects among consumers. To overcome such a problem, an attempt was made to formulate herbal shampoos, which can be used as an alternative to synthetic ones. The present work focused on the formulation and evaluation of herbal shampoos using some traditional medicinal plant extracts that are readily available and used for medicinal purposes across Ethiopia. Results revealed that the formulated herbal shampoos showed acceptable properties when assessed for their quality parameters. The pH values were in a range 5.90 ± 0.22-7.45 ± 0.19, and lower surface tension (23.39 ± 0.20-31.89 ± 1.04 dyne/cm) together with acceptable viscosity with good shearing properties were obtained. The formulated products F-1 and F-4 showed good quality in terms of tested parameters, were stable, and had antimicrobial activities. The surface morphology studies of formula F-4 using SEM showed a clear and smooth surface, ensuring the cleansing and conditioning performance. This result was comparable to the marketed shampoo. Therefore, the formulated shampoos can be used as an alternative to synthetic ones; however, additional evaluation together with further research and development are required to ensure better quality and safety.