Formulation and antimicrobial evaluation of polyherbal anti-dandruff shampoo along with pharmaceutical analysis

Introduction

Scalp infection is one of the major causes of hair damage caused by various pathogens like bacteria, fungus, virus or parasites. Among these, the most common pathogens involved in hair infections are bacteria and fungus. Bacterial infection of hair follicles (folliculitis), the infestation of head lice (Pediculosis capitis), and fungal infection of scalps such as ringworm (Tinea capitis) and Piedra are common diseases of scalps and hairs. Itching and excessive flaking of the scalp are seen with dandruff (Seborrheic dermatitis). The shampoo is most widely used as cleansing and soothing agent for hair nourishment and ‎protection from various bacterial and fungal infections. There are so many products are ‎available in the market labelled against various disorders of hair. But the continuous use of ‎shampoo cause dryness, fall and loss of lustre of hair, irritation of scalp and eye, due to ‎synthetic foaming and cleaning surfactant which are being added in the shampoo. To ‎overcome these problems some medicinal herb based shampoos along with natural additives ‎have been developed in which extract of herbs were used as ingredient for safe and effective ‎treatment against fungal and microbial infection of scalp and hair care [1].

In view of above the present study has been envisaged to design and develop an antifungal shampoo based on natural ingredients and additives. The herbs are selected which are described in classical Unani literature to nourish, protect and exhibit antimicrobial and antifungal activities substantiated with recent scientific studies such as Aamla (Phyllanthus emblica L.) helps in skin protection and promotes hair growth. Other properties are antifungal, antimicrobial, radio protective, anti-oxidative [2, 3]. Some recent studies demonstrated significant antimicrobial activity [4, 5]. Gul-e-Gurhal (Hibiscus rosa sinensis L.) has anti-greying properties and promotes hair growth ‎also [6, 7]. Persiaoshan (Adiantum capillus-veneris L‎.) is utilized in remedies for hair, scalp and beneficial in alopecia patches [8]. It also helps in hair growth and maintaining healthy and long hair [2]. Kalonji (Nigella sativa L.‎) demonstrated antifungal, anti-bacterial, anti-inflammatory, antioxidant activities [9, 10]. Hulba (Trigonilla foenum-graecum L.) has been used traditionally for various pharmacological ‎effects, such as anti-diabetic, anti-cancer, anti-fungal, anti-pyretic, anti-bacterial and anti-‎oxidant‎ [11]. Besides, the seeds of Hulba have been used as anti-lice, anti-dandruff and are used for hair growth and soothing effects [12]. Ethanol extracts of leaves of Hulba had significantly higher growth promoting effects compared to standard, i.e., minoxidil [13]. Neem (Azadirachta indica A.Juss) exhibits antimicrobial, antifungal, anti-inflammatory activities, used to kill the lice and various infectious disorders of scalp [14, 15, 16, 17]. Further the study has been subjected to physico – chemical standardization for the reproducibility and the antimicrobial and antifungal studies were also carried out to validate efficacy of the prepared shampoo.

Materials and methods

The present study aimed to design and development of antidandruff shampoo based on Unani herbal drugs along with standardization and evaluation of antimicrobial and antifungal activities. The ingredients of shampoo were seeds of Azadirachta indica A. Juss, Nigella sativa L., and Trigonella foenum graecum L., fruits of Phyllanthus emblica L.‎, leaves of Adiantum capillus-veneris L., and flower of Hibiscus rosa sinensis L.

Procurement of herbal drugs and other necessary ingredients of shampoo

All the ingredients Unani anti-dandruff shampoo collected by the authors themselves directly from plants except Persiaoshan (Adiantum ‎capillus – veneris) and identified by the pharmacognosist. Persiaoshan was procured from the Dawakhana ‎Tibbiya College, a herbal pharmacy unit of Aligarh Muslim University, Aligarh. The specimens have been submitted in the museum of the Department of Saidla and voucher no.301/8 issued.

Preparation of Extract

First, all the drugs mentioned in table 01 were cleaned and washed from impurities, if any. After cleaning, crude drugs were dried in an oven at 40◦C except Gul-e-Gudhal (Hibiscus rosa sinensis), which was dried in shed, and then the coarse powder was made by grinding of all the crude drugs in equal quantity. The powdered formulation was extracted by the Soxhlet’s apparatus using double distilled water as a solvent for 06 hours. These extracts were dried on the water bath at a controlled temperature and stored well for use.

Method of preparation

Antidandruff shampoo from Unani drugs was prepared by the method of [18, 19]. First, extract was dissolved in distilled water at 75◦C and homogenized properly. In order to ‎formulate shampoo, additives or excipients shown in table 02 were added one by one to the ‎extract solution. During addition of other ingredients, the extract solution was continuously ‎stirred and heated on the magnetic stirrer equipped with a hotplate. The solution ‎ stirred continuously till homogenization. Different batches (F1, F2 and F3) of shampoo were ‎prepared by the same method by optimizing the concentration of extract and Cocamidopropyl ‎betaine, as shown in table no. 02.‎

Physico-Chemical Evaluation of Herbal Anti-dandruff Shampoo

To identify the best batch all the prepared batches were tested for organoleptic properties, pH, and percentage of solid residue. Based on the results of these tests, the F1 batch of the prepared formulation was found suitable and is the best-optimised formula. Therefore, F1 was selected for further evaluation and stability studies. All the tests were performed on in house formulations and were executed in triplicate [20, 21, 22].

Organoleptic characteristics

The prepared formulations evaluated for the colour, odour, clarity and transparency, foam forming property, physical stability, fluidity and viscosity by the method of Badi and Khan  2014andKrunali et al. 2013 [23 ,24]. The parameters observed for physical stability, homogeneity and phase separation for 02 days.

pH of 10% shampoo solution

The 10% shampoo solution was prepared in distilled water and pH was determined at 25◦C with the help of digital pH meter [25].

Determination of solid residue in shampoo:

4gm of shampoo was taken in a preweighed, clean, and dry petri dish. The dish was kept on water-bath to evaporate the liquid portion of shampoo completely. The weight of the petri dish contains shampoo was measured till it reaches the constant. Then the weight of the solid residue of shampoo was calculated in percentage [20, 26].

Wetting time of Shampoo

A canvas sheet was taken and cut into discs having 1-inch diameter weighing 0.44gm each. 1%w/v shampoo solution was prepared in a beaker. Disc pieces were dropped over the shampoo solution and allowed to sink. The stopwatch was started immediately after dropping the discs, and the exact time was noted at the beginning of the sinking of canvas pieces [22, 23, 24].

Detergency evaluation

It was determined by the method of Thompsonet al. 1985 [27], with slight modifications. Hair tresses were obtained from local hair saloon and artificial sebum was prepared according to the formula shown in table 03.

Hair samples were washed with a 5% SLS solution (Sodium Lauryl Sulfate) and dried. After divided into hair-swatch of 5gm, each placed in 20ml of 10% artificial sebum solution in hexane for 15 minutes. The hair-swatch was removed from solvent, evaporated at room temperature and weighed to determine the sebum load. The soiled hair swatches were cut into half equal samples of 2.5gm each; one part of each swatch was kept as control, and the other was washed with a shampoo solution. The shampoo solution of 10% w/v i.e. 200ml distilled water and 20gm of shampoo was prepared in a flask. The test samples soiled with sebum were put in the flask containing shampoo solution, and the temperature of the water was maintained at 35◦C by putting it on a hot plate. The flask was shaken for 4min at a rate of 50 shakes per min. Now the hair samples were taken out from the solution, dried and weighed till constant. The amount of sebum removed was calculated by using the following equation

DP = 100 – (T×100/C)

Where DP is the percentage of detergency power

C is the weight of sebum in the control sample

T is the weight of sebum in the test sample

Surface Tension

Surface Tension of shampoo was measured using a stalagmometer.  The test was carried out with a 10% shampoo solution in distilled water at room temperature [23, 26, 28].

Viscosity

It was determined by using Ostwald Viscometer at room temperature [29, 30].

Foam volume and foam stability

It was calculated by using the Cylinder shake method. It is an easy and widely used technique for foam volume evaluation because of its fast results and reproducibility.  In a 250ml graduated cylinder, 50ml of 15% shampoo solution was taken and clogged with the stopper. The cylinder was shaken 10 times in an upward and downward direction. The total volume of foam formed was recorded immediately. For foam stability the foam volume was observed for 4 minutes, and recorded at 1, 2, 3 and 4 minutes [31, 32].

Dirt dispersion

10 ml distilled water with two drops of shampoo were taken in a 25 ml graduated test tube. One drop of India ink added to this solution. The tube was closed using stopper and then shaken ten times. The amount of ink in the foam was estimated as None, Light, Moderated and Heavy [31, 33].

Thermal Stability

Six samples of shampoo were taken in different glass tubes. Three were placed in an oven at 45⁰ C and rest in the chiller at 5⁰ C. After one week, their appearance and physical stability were inspected [19].

Mechanical Stability

Mechanical stability assessed by the centrifuge method. 04 samples of shampoo were taken in centrifuge tubes and centrifuged at 2400 rpm for 3 minutes. The structural and physical stability were observed [19].

Antimicrobial Evaluation

The antibacterial and antifungal activity of the test shampoo was also evaluated for analyzing its antimicrobial properties especially against Malassezia furfur the main causative organism of dandruff properties by the following method. This test was outsourced by the department and conducted by the Mahesh Azyme Biosciences, Bengaluru, Karnataka.

Antibacterial activity: Antibacterial efficacy of herbal Anti-Dandruff shampoo was assessed by Micro titer plate method (96 wells) to check the Minimum Inhibitory Concentration (MIC) against gram negative bacteria strains (Pseudomonas aeruginosa, Proteus vulgaris), and gram positive bacteria strains (Staphylococcus aureus and Staphylococcus epidermidis) [34]. Tetracycline was used as a standard drug.

Sample was prepared by dissolving 10mg test drug in 1mL Dimethyl sulfoxide (DMSO) and 100µL of the dissolved sample was used to check the Minimum Inhibition Concentration (MIC) for particular organisms. Luria Bertani (LB) broth (tryptone 10g, sodium chloride 10g, yeast extract 6g, and distilled water 1000mL) 100mL was prepared and autoclaved at 121°C for 15 min used as media.

Plate preparation: 300µL deionized water was added into the surrounding wells samples of micro titre plates (A₁ to A₁₂, B₁₂ to H₁₂, H₁₁ to H₁, and G₁ to B₁) to prevent the sample from drying. 100µL sterilized LB broth was added to all the remaining wells. 100µL of 0.1% resazurin dye in well B₂ to E₂ was added as colour blank. In well B₃ to E₃ test organisms and 100µL of 0.1%, resazurin dye was added as culture control. 100µL of the test formulation added in well B₄ to E₄ and serially diluted by transferring 100µL of the mixture to subsequent wells up to 11^th well and 100µL of the excess sample was discarded from 11^thwell as shown in figure 02 (a). Another plate was prepared for standard drug i.e. Tetracycline and instead of test formulation 100µL of the standard drug added in the well B₄ to E₄ and serially diluted by transferring 100µL of the mixture to subsequent wells up to 11^th well and 100µL of the excess sample was discarded from 11^thwell as shown in figure 02 (b). 100µL of culture was inoculated to serially diluted sample (test formulation and standard drug) wells. Pseudomonas aeruginosa from B₄ to B₁₁, Proteus vulgaris was inoculated to wells from C₄ to C₁₁, Staphylococcus aureus was inoculated to wells from D₄ to D₁₁, Staphylococcus epidermidis was inoculated to wells from E₄ to E₁₁ and 100µL of 0.1% of resazurin dye was added to all the test sample wells.  The plates were incubated at 37° C for 24h.

Anti-fungal activity: Herbal shampoo was also analyzed for its antifungal activity against Malassezia furfur, a causative organism for dandruff.  To check the minimum inhibitory concentration of the herbal antidandruff shampoo, well diffusion method was used [35]. For sample preparation 10mg of the test formulation and positive control Fluconazole was dissolved in 1mL of DMSO (Dimethyl sulfoxide) in sterilized eppendorf respectively. From this stock solution, different samples were prepared of 100µg, 200µg, 300µg and 400µg concentration. Potato Dextrose Agar (PDA) was used as culture media. 100µL inoculums of Malassezia furfur added into the PDA plates and spread thoroughly. On agar plates, five wells measuring 5.5 mm were made and loaded with 50µL of the different samples in respective wells. DMSO 50µL added in the middle well treated as control. The culture plates were incubated at 25°C for 72 hours. The zone of inhibition was recorded in millimeters.

Accelerated Stability Studies of Herbal antidandruff shampoo

Accelerated Stability Studies of the shampoo was done according to The European Cosmetic, Toiletry and Perfumery Association (COLIPA) and Consumer Healthcare Products Association (CHPA) guidelines. The stability chamber was maintained at a temperature of 40±2⁰C with 75±5% relative humidity for creating stress conditions for the formulated shampoo for a minimum time period of 03 months predicting a shelf life of up to 24 months  [36]. The parameters assessed to evaluate the shelf life and physicochemical changes were organoleptic properties and physicochemical characteristics such as pH, viscosity, dirt dispersion, foam stability, detergency evaluation, thermal stability and, mechanical stability etc. These parameters were evaluated at 0 (initial), 1, 2 and 3 months. These tests are done to assure the physical integrity of the test product under appropriate conditions of storage, transport and during use. The container closure system is of 100ml capacity bottles, made up of transparent polyethylene terephthalate (PET). About 75gm of formulated Unani antidandruff shampoo was filled into the bottles and closed with white polypropylene threaded flip cap. All precautions were taken while packaging these samples into the containers. The bottles were labeled properly, including formulation name, date of preparation, date of commencement of thermal/humidity challenge, date of withdrawal etc. 03 packed samples were placed in temperature and humidity control cabinet for accelerated stability studies and tested one by one at the end of 01, 02, and 03 month respectively [36, 37].

Results and Discussion

Optimization of the Batch

To optimize the best formulation among different batches of Unani Anti-Dandruff shampoo (F1, F2 and F3) based on herbal drugs, organoleptic characters, pH and percentage of solid residue was assessed. The general outward appearance, fragrance and clarity of the shampoo have the first impression on consumers. All the batches of shampoo were clear, have soothing and pleasant odour and brown in colour except batch F2 and F3 which were dark brown in colour [32]. Although foam generation (lathering) has no correlation with the cleansing ability of shampoos, it is of paramount importance to the consumer and is therefore an important criterion in evaluating shampoos. Results showed that the tested shampoos in distilled water have good foam formation with desired viscous fluidity except batch B2 which was watery in nature  [38]. Shampoos showed stability, homogeneity and no phase separation when observed for two days. A good shampoo must have neutral or slightly acidic pH, ranges between 5.0 to 7.0. Shampoo pH level is responsible for improving and enhancing hair quality, minimizing eye irritation, and stabilizing the scalp’s ecological balance. Mild acidity prevents swelling and promotes tightening of the scales, thereby inducing shine. Thus, the current trend is to promote shampoos with lower pH as a way to minimize hair damage. Batches F1 and F2 of the shampoo are slightly acidic in nature showed pH ‎6.8‎ and 6.5 respectively. While the pH of batch F3 was found to be neutral i.e. ‎07 [38, 39].‎ Usually, the percentage of solid materials in good shampoo remains between 20-30% [40, 41]. Percentages of solid contents of all the batches of Unani antidandruff shampoo were found within the given range and batch F1 depicted least ‎percentage of solid residue i.e. 20.103%. Based on these results as depicted in table 4 batch F1 was found most suitable and rest of the tests like physico-chemical and antimicrobial studies were carried on it. Figure 1 presents the image of batch F1 of Unani Anti-Dandruff shampoo.

Physico-chemical evaluation

Wetting phenomena are complex and depend upon several processes and factors such as diffusion, surface tension, concentration, and the nature of the surface being wetted. Each wetting agent has to reduce surface tension [38]. The tested shampoo had acceptable ranges of wetting times and was found to be 140.93sec. The prime function of any shampoo is its detergency action over the removal of sebum or any dirt deposited on the hair. Previous researches revealed that a good shampoo should possess detergency power between 55 to 65 % [41, 42]. Higher values of detergency may strip off the natural oils of the hair and lower detergency power does not exhibit a good cleaning action. Detergency power of formulated Unani anti-dandruff shampoos was found to be 60%, hence considered being of good and suitable detergency power. Surface tension of shampoo depends on the quantity of surfactant present. The lesser the surface tension, the stronger the cleaning ability of the shampoo [38]. A reduced surface tension affects positively on the cleansing ability of the shampoo in two ways: first by facilitating the spread of the shampoo solution and increasing its wetting ability for the surface and second by facilitating the removal of dirt from hair by keeping it in suspension. A good shampoo should be able to decrease the surface tension of pure water from 72.28 dyn/cm to about 40 dynes/cm [43]. A reduction from 90.8 dynes/cm to 32.2 dynes/cm by the Unani anti-dandruff shampoos is an indication of their excellent detergent action. Product viscosity plays an important role in defining and controlling many attributes such as shelf-life stability, product aesthetics such as clarity and ease of flow of the product, spreading ability of shampoo on the hair, and product consistency in the packaging [38]. Those shampoos having viscosity below 1000 cps tend to be runny; and if the viscosity is over 2000 cps, it may not provide good pourability. The viscosity of formulated Unani anti-dandruff shampoo was found to be 1486.4cps at room temperature.  Dirt that remains in the foam will be difficult to rinse away and it will get redeposit on the hairs. Thus, the dirt should stay in the water portion for achieving better cleansing action. Dirt dispersion test of formulated Unani anti-dandruff shampoo showed that the ink distribution found to be ‘none’ in foam, thus, the shampoo has good cleansing action [23]. The foam has the paramount importance to the consumer and is therefore Foamvolume ‎and ‎stabilityan important criterion for shampoo evaluation. For a good shampoo foam should be 100 ml or more. Foam volume of tested shampoo was good and found to be 186ml which reduced to 178ml in 05 min shows good stability also [38]. There were no physical changes appeared in the shampoo during the thermal and mechanical tests. Thus, the formulated Unani anti-dandruff shampoo was found to be thermally and mechanically stable [19]. All the results are depicted in table 5.

Antimicrobial studies of herbal antidandruff shampoo

Polyherbal Unani antidandruff shampoo showed inhibition against tested gram-positive and gram-negative bacterial strains as shown in table 6 and figure 2 (a and b). Screening of medicinal plants for antifungal activity is also important for novel drug discovery. Shampoo was also tested against the, dandruff causing fungus, Malassezia furfur andthe results suggested noteworthy antifungal activity of the test formulation and was also found greater than the standard drug Fluconazole (table 7). The diameter of zone of inhibition increased as the concentration of the shampoo increased, showing a dose-dependent effect (figure 2 c and d) [44].

 Stability studies of herbal antidandruff shampoo

Stability testing is to be performed to provide evidence on how the quality of a drug substance varies with time under the influence of various environmental factors such as temperature, humidity, and light, and hence to establish shelf life under recommended storage conditions [45]. Accelerated conditions are to predict tentative shelf life for a new or modified product with a short span of testing under stress conditions. As per CHPA guidelines accelerated testing storage conditions with a minimum time period of 03 months predicting a shelf life of up to 24 months. Under accelerated storage condition, 5% change in the values of parameters from the initial values denotes significant degradation in the formulation [36]. According to the results depicted in the table 08‎, there were no considerable changes found in the organoleptic charachters and physico-chemical properties of the test formulation. Thus, the quality of test shampoo developed from natural ingredients was remains good under stressed condition, during the test period and the shelf life was predicted as at least of 02 years.

Conclusion

A polyherbal stable and effective Unani anti-dandruff shampoo based on traditional knowledge was formulated without any synthetic drug. The results showed that the formulation “F1” of antidandruff Unani shampoo contains all good characters of an ideal shampoo. In-vitro antimicrobial studies specifically against Malassezia furfur, a causative fungus for dandruff confirm the antidandruff ability of the tested shampoo. The shelf life of shampoo was found as 02 years under normal storage conditions. Further, the manufacturing procedure of the batch F1 of Unani antidandruff shampoo can be used for further reference and reproducibility. This shampoo may be the best replacement for synthetic counterparts, if formulated with best quality ingredients in optimized combinations. In addition, published data of its ingredients also suggested that the above mentioned herbs have anti-graying, anti-hair fall properties which may further subjected to experimental and clinical trial.

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