I[NTRODUCTION]{.smallcaps} {#sec1-1}
==========================

Shampoo is a cosmetic product used to clean hair and scalp, and contains several types of constituents in its formulation such as surfactants, solvents, coloring agents, pH adjustments, binders, preservatives, and so on.\[[@ref1]\] The word *shampoo* was derived from the Indian word *cāpnā* meaning "push," which was extended to indicate the action of washing hairs.\[[@ref2]\] Shampoos available in markets exist in various forms such as powder, liquid, lotion, cream, jelly, and aerosol.\[[@ref3]\] Some ingredients may be added to shampoos to specialize their application, for example, conditioning shampoo, antidandruff shampoo, baby shampoo, beauty shampoo, and so on.\[[@ref2]\] Herbs have been used from ancient times for hair treatment and fortification.\[[@ref4]\] Plants are rich in bioactive constituents including vitamins, essential oils, amino acids, hormones, enzymes, and so on, which make them an appropriate choice for using in cosmetics.\[[@ref5]\] There are several medicinal herbs that are used to produce herbal shampoos available in the market.\[[@ref3]\] The use of herbal shampoos has increased over the past decades. Consumers believe that herbal shampoos are safe and harmless.\[[@ref5]\] Herbal shampoos available in the market contain herbal extracts at the base of synthetic constituents, but crude, powder, or other derivative forms of plants may also be used in formulation.\[[@ref5][@ref6]\] Formulating a shampoo using completely natural constituents is a troublesome project.\[[@ref7]\] *Glycyrrhiza glabra* (*G. glabra*) is a perennial plant belonging to the family *Fabaceae*. The term *licorice* originated from the Greek word *glykosrhiza* in which *glykos* means "sweet" and *rhiza* means "root." Licorice would be ready to be unearthed at the end of the third season but it is usually harvested in the autumn of the fourth year.\[[@ref8]\] It is indigenous to the Mediterranean region and central and southwestern Asia, especially Iran, and cultivated in the temperate and subtropical areas.\[[@ref9]\] In common English, *G. glabra* is mentioned using different terminologies: licorice, common licorice, licorice root, liquorice, sweet licorice, sweetwood, drop, licorice powder, liquorice plant, Spanish juice-root, Spanish juice-wood, and Spanish liquorice.\[[@ref10]\]

The rhizome and root of *G. glabra* have been used for different medicinal and economical purposes. They have various applications in cosmetics, medicine, and pharmaceutical industries, and are used in confectionary, food, and tobacco industries as a sweetener.\[[@ref11]\] *G. glabra* is reported to possess varieties of biological properties such as antiviral, anti-inflammatory, antioxidant, hepatoprotective, antibacterial, cytotoxic, antiprotozoal, and antitumor, and has been utilized in various clinical studies such as gasterointestinal, endocrinological, dermatological, and respiratory diseases.\[[@ref12]\] *G. glabra* contains varieties of phytoconstituents such as sponins (mainly glycyrrhizin), flavonoids (mainly liquiritigenin and liquiritin), isoflavons (mainly glabridin, coumarins, stilbenoids), and miscellaneous compounds.\[[@ref12]\] This study was planned to formulate a herbal shampoo (licorice shampoo) using the extract of *G. glabra* in order to introduce a product with defined standard characteristics. Licorice was choose licorice was based on its application in cosmetics; its anti-inflammatory, antibacterial, and antifungal activities; and the existence of saponins in its phytoconstituents, which makes it an appropriate choice for a shampoo product.

M[ATERIALS AND]{.smallcaps} M[ETHODS]{.smallcaps} {#sec1-2}
=================================================

All the materials were purchased from authentic companies: Dehyton® from BASF Co., Germany; sodium chloride, triethanolamine, glycyrrhizic acid standard, and methyl and propyl paraben from Merck Co., Germany; methyl paraben (Nipagin® M) from Clariant, Switzerland; and sodium lauryl ether sulfate (SLES; Texapon® N70) from Cognis, Düsseldorf Co., Germany. The commercial shampoo was purchased from Darugar Co., Iran.

Preparation of plant and extraction {#sec2-1}
-----------------------------------

The root of *G. glabra* was collected from Sepidan region, in Fars Province, Iran, and the scientific name was approved by Dr. Mohammad Azadbakht (PhD of Pharmacognosy, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran). The root was cleaned, dried, and pulverized. 100 mg of root was soaked in 300 ml boiled water for 48--72 h. It was then subjected to percolation extraction method with boiled water. After extraction of 300 ml solution, ammonia was added to the solution up to pH = 8 and then boiled to reduce the volume to 150 ml; the solution was then filtered and concentrated to 75 ml.\[[@ref13]\]

HPLC analysis {#sec2-2}
-------------

The assay of extract base of the glycyrrhizic acid was performed by high-performance liquid chromatography (HPLC) apparatus (Knauer-YF 50-C18, 100A column \[250 mm × 4.6 mm with C18 precolumn\] plus quaternary gradient pump \[pu-2089\] plus UV-E4310 intelligent UV/vis detector).

Sample preparation: 0.1535 mg of dried extract was adjusted to 100 ml by using ammoniac 2%. Standard: 5 ml of standard glycyrrhizic acid solution (0.625 mg glycyrrhizic acid in 25 ml ammonia 2%) was adjusted to 100 ml by ammonia 2%. The sample was eluted with isocratic mobile phase consisted of acetic acid 2.5% and ammonia HPLC grade at the flow rate of 1 ml/min with 10 min retention time (RT). All procedures were performed in triplicate.

Formulation of herbal shampoo (licorice shampoo) {#sec2-3}
------------------------------------------------

The formulation procedure was executed at the temperature of 70°C. Texapon® and Dehyton® (pH regulator) were mixed and the extract was added, followed by addition of a mixture of methyl and propyl paraben (preservatives) in water, 1.3 g of salt, 1.6 g of cocamide diethanolamine (CDE; foaming agent), and 0.7 g of salt. The temperature was then reduced to 45°C, and *Citrus aurantium* essential oil in 0.9 g of CDE was added \[[Table 1](#T1){ref-type="table"}\]. Finally, the mixture was eluted with water to the final volume. Similarly, as a control test, the nonherbal shampoo without the extract was also tested.

###### 

Composition of formulated base shampoo

![](JPBS-10-208-g001)

Evaluation of formulated and commercial shampoo {#sec2-4}
-----------------------------------------------

Visual inspection, physicochemical controls, conditioning performance tests,\[[@ref14]\] and microbial control evaluation were done to evaluate the quality of new formulation.

### Physical appearance/visual inspection {#sec3-1}

The formulated shampoo was evaluated for physical characteristics including color, clarity, and odor.\[[@ref15]\]

### Determination of pH {#sec3-2}

The pH of 10% solution of formulated shampoo in distilled water was measured using a pH meter at room temperature.\[[@ref16]\]

### Determination of the amount of solid contents {#sec3-3}

A sample of formulated shampoo was kept at a temperature of 105°C for 3 h and weighed. The measured weight is the amount of solid contents.\[[@ref17]\]

### Cleansing evaluation {#sec3-4}

Cleaning ability factor was determined by gravimetric method.\[[@ref18]\] Briefly, a wool skein was weighed and set in CCl~4~; it was then cleaned, dried, weighed again, and dipped with standard soil of the American Society of Testing Materials (ASTM), then weighed and washed in a pan containing 2% shampoo solution for 5 min with the speed of 60rpm, followed by rinsing, drying, and weighing. Detergency yield was calculated using the following equation:

![](JPBS-10-208-g002.jpg)

**Equation 1**. W~1~: primary weight of skein, W~2~: weight of soil-dipped skein, W~3~: final weight of skein

### Foaming ability evaluation {#sec3-5}

Foaming ability was determined using Ross--Miles method: 150 ml of licorice shampoo was poured on 25 ml of aforementioned prepared shampoo in a graduated cylinder from a height of 90 cm, and then the volume of created soap was measured.\[[@ref19]\]

### Wetting time test {#sec3-6}

Based on Draves method, a 5 g cotton skein connected to a 2.5 cm cotton string was dropped in a cylinder containing 500 ml of 2% shampoo at a temperature of 45°C. As the string got wet, it separated. The time from dropping the skein till the string isolation was considered as the wetting time.\[[@ref20]\]

### Viscosity profile {#sec3-7}

Pointer viscometer was used at room temperature to determine viscosity. The samples were placed in a sample container and allowed to settle for 5 min, and the viscosity measured a rotating speed of 50 rpm.

### Microbial control assessment {#sec3-8}

Soybean-casein Digest Agar Medium (SCDA) was used for preliminary evaluation of microorganisms. Microbial suspension was also prepared at concentrations of 10^5^ and 10^8^ for strains of *Candida albicans, Escherichia coli, Pseudomonas aeroginosa*, and *Staphylococcus aureus* a sample of shampoo was diluted with SCDA at a ratio of 1:10; then 1 ml of the prepared diluted solution and 9 ml of SCDA was added into four test tubes. 0.1 ml of each microbial suspension was added individually to one of the pertinent test tubes. The test tubes were sealed, shaken, and kept at a temperature of 37°C for 24h.\[[@ref21]\]

***Microbial limits test*** This test was carried out based on plate count method: 1 ml of 1:10, 1:20, 1:30, 1:40, and 1:50 of diluted licorice shampoo was added separately into five test tubes containing 9 ml of SCDA. Then 1 ml of the mixture of each test tube was put on a plate, and added by 15 ml of nutrient-agar and melt agar then kept in an incubator for 24 h.\[[@ref21]\]

***Antimicrobial preservative effectiveness test*** 20 ml of licorice shampoo and 0.1 ml of each microbial suspension were added to individual pertinent test tubes and kept at room temperature for 7, 14, 21, 28 (and 35 for *C. albicans*) days. 1 ml of the contents from each tube was taken in two sterile plates and then 15--20 ml of 40°C melt soybean-casein digest agar was added to it. After solidifying, they were kept at 37°C for 24 h.\[[@ref21]\]

### Evaluation of consumer satisfaction {#sec3-9}

The study protocol was approved by the Institutional Ethical Committee (Nom:78.5) at Shiraz University of Medical Sciences. The licorice shampoo was given to 50 volunteers to be used for 30 days. The volunteers were aged 25--35, living locally, instructed on shampoo usage, and recommended not to use any other shampoos during the trial period; at the end of the trial, the volunteers were asked to fill up a questionnaire. The questionnaire was designed to comprise several aspects related to the shampoo, and it included questions on the quality of shampoo, effect on hair style, hair loss, scalp, and eye irritation. The consumers were also asked about the impact of shampoo on inducing or revealing hair complications including itching, dandruff, trichoptilosis, and spout. Furthermore, the consumers were asked to rate different aspects of the shampoo as excellent, good, or bad.

S[TATISTICAL]{.smallcaps} A[NALYSIS]{.smallcaps} {#sec1-3}
================================================

The results were represented as mean ± standard deviation. The data were analyzed and compared using SPSS software version 20 via analysis of variance followed by student's *t*-test.

R[ESULTS]{.smallcaps} {#sec1-4}
=====================

Extract standardization {#sec2-5}
-----------------------

The RT of glycyrrhizic acid was recorded as standard at 5.27 min and 5.28 min for the extract. The content of glycyrrhizic acid in extract was quantified 18.39% which has been shown in [Figure 1](#F1){ref-type="fig"}.

![The HPLC peak of glycyrrhizic acid as standard; retention time (RT): 5.27 (left) and the HPLC peak of *G. glabra* watery extract; RT: 5.28 (right)](JPBS-10-208-g003){#F1}

Evaluation of shampoos {#sec2-6}
----------------------

The results of quality assessments on licorice, nonherbal, and commercial shampoo are explained in [Table 2](#T2){ref-type="table"}.

###### 

Physicochemical assessment of formulated and commercial shampoos

![](JPBS-10-208-g004)

### Physical appearance/visual inspection {#sec3-10}

The colors of licorice and commercial shampoo were dark brown and yellow, respectively. The viscosity, fluidity, and odor were acceptable in comparison with commercial shampoos.

### pH {#sec3-11}

The pH of commercial, nonherbal, and licorice shampoos at 45°C was 7.34, 8.33, and 8.70, respectively.

### Percentage of solid contents {#sec3-12}

The percentage of solid contents in the commercial, nonherbal, and licorice shampoos was 62%, 57%, and 81%, respectively.

### Cleansing ability {#sec3-13}

The cleaning yield of nonherbal (97%) and licorice shampoos (90%) were significantly higher than the commercial shampoo (58%) at 45°C (*P* \< 0.05).

### Foaming ability {#sec3-14}

The volume of created foam at 45°C was 120, 147, and 150 ml for commercial, nonherbal, and licorice shampoos, respectively, without significant differences (*P* \> 0.05).

### Wetting time {#sec3-15}

The wetting time of commercial, nonherbal, and licorice shampoos was 17, 18, and 18.2 s, respectively based on Draves method, and there was no significant difference (*P* \> 0.05).

### Viscosity {#sec3-16}

The viscosity of commercial, nonherbal, and licorice shampoos were 55, 220, and 15 cp, respectively. The viscosity of licorice shampoo was significantly lower than commercial and nonherbal shampoos (*P* \< 0.05).

### Microbial control assessment {#sec3-17}

***Primary microbial assessment*** The results of primary assessment indicated no microbial growth in the diluted samples.

***Microbial limits test*** No microbial growth was observed during the study of the prepared herbal shampoo.

***Antimicrobial preservative effectiveness test*** The samples studied for microbial growth were prepared after appointed days. No sign of microbial growth was observed.

### Consumer satisfaction {#sec3-18}

Of the 50 volunteers, 47 had filled the assessment questionnaire. The types of hair of the consumers are shown in [Table 4](#T4){ref-type="table"}. The results of the analyses of effectiveness of and customer satisfaction with licorice shampoo are described in Tables [5](#T5){ref-type="table"}--[8](#T8){ref-type="table"}. Most of the consumers (83%) stated no hair loss effects while using the licorice shampoo, whereas 17% had the opposite opinion. Most of the consumers were satisfied with the efficacy, quality, and safety of the licorice shampoo \[[Table 3](#T3){ref-type="table"}\].

###### 

Consumer reports on the quality of licorice shampoo

![](JPBS-10-208-g005)

###### 

Consumer reports on hair style after using licorice shampoo

![](JPBS-10-208-g006)

###### 

Consumer reports on the quality of licorice shampoo based on gender

![](JPBS-10-208-g007)

###### 

Consumer reports on hair loss after using licorice shampoo

![](JPBS-10-208-g008)

###### 

Consumer reports on elimination of hair and scalp problems after using licorice shampoo

![](JPBS-10-208-g009)

###### 

Consumer reports on scalp complications and eye irritation after using licorice shampoo

![](JPBS-10-208-g010)

D[ISCUSSION]{.smallcaps} {#sec1-5}
========================

In this research, herbal shampoo was formulated by applying the aqueous extract of *G. glabra*. Shampoos are used mainly for cleaning, which is determined principally by its surfactant detergency property.\[[@ref22]\] *G. glabra* is rich in saponins, which are commonly known as natural surfactants possessing good foaming and detergency ability.\[[@ref12][@ref23]\] Texapon® N70 is an anionic surfactant used as a detergent and foaming agent.\[[@ref24]\] Anionic surfactants are the more popular surfactants, which may be due to the following reasons: hair charge is negative, and consequently, anionic detergents absorb the positively charged oils that bind to the hair and cuticle, and as a result, the surfactant can be washed off easily.\[[@ref22]\] Lauryl sulfates are the chief detergents as they produce ample foam, work well in soft and hard water, and washes off well.\[[@ref25]\] Cocamide diethanolamine is a nonionic surfactant used as foam stabilizer and foam booster, and for increasing viscosity.\[[@ref26]\] Dehyton® is an amphoteric surfactant used as a foaming agent.\[[@ref24]\] Commercial shampoos comprise 15--40% of detergent surfactants.\[[@ref25]\]

The viscosity of the formulation escalated through reducing the interval of micelles particles, such a delicate aggregation which is accomplished by depression of anionic surfactants density, regulated by the sodium ion concentration.\[[@ref26][@ref27]\]

The triethanolamine is used as a basic agent to counteract the acidity effect of the glycyrrhizic acid contained. Furthermore, the aforementioned base is capable of regulating the formulation viscosity.\[[@ref28]\] As shampoos are a highly used cosmetic, they must conform to the standards. The herbal shampoo was evaluated by visual inspection, physicochemical evaluations, microbial control assessment, and consumer polls. The dark brown color of the formulated herbal shampoo originates from the licorice extract; using natural dyes during formulation could improve the appearance of the product. Management of pH in shampoos has significant effects on the hair and scalp qualities and prevents eye irritation.\[[@ref29]\] The pH range of most commercial shampoos in Brazil was reported to be 5.5--7; however, no standard range for shampoo pH has been defined yet.\[[@ref30]\]

The solid content in shampoos has an effect on rinsing. Shampoos with low amount of solid content can be washed off quickly, and conversely, high amount of solid content in shampoo makes it harder to wash off.\[[@ref31]\] Cleaning ability of the shampoo is a very important factor in its evaluation. A high amount of detergents dries the hair by eliminating natural oil.\[[@ref32]\] Licorice shampoo has shown excellent cleansing in comparison with commercial ones.

The foaming capability of the product is considered to be an element of public desirability toward the merchandise. It is believed that the higher the foaming obtained, the better the quality of the shampoo. This belief is exceptionally inconsistent with the reality experienced within the scientific community.\[[@ref33]\] The foam stability of licorice and commercial shampoo was similar; however, higher foam was produced in licorice shampoo due to high amounts of saponins in the extract. The wetting time of the surfactant depends on the concentration used.\[[@ref34]\] As the wetting time increases, the detergent completely envelopes the soil and dissolves better. The wetting time durations in the commercial, nonherbal, and licorice were not significantly different. The ideal rheological characteristics of shampoo are effective on shelf life, package consistency, flow ability, spreading, and clarity.\[[@ref34]\] Microbial contamination in pharmaceutical products would cause harm to the health of the consumer and may affect the physicochemical properties of the product including color, odor, consistency, and so on. Consequently microbial controls are necessary for product evaluation.\[[@ref35]\] Antimicrobial preservative effectiveness test is an important phase in the good manufacturing practices to evaluate the biological activity of preservatives.\[[@ref21]\]

Previously, the antimicrobial activity of licorice extract had been proved, and thus, it can have a synergy effect with preservatives. Similarly to the commercial shampoo, licorice shampoo was free of microbial contamination at all stages. According to the reports from consumers, 74.5% were satisfied with the foaming and dispersing ability of the shampoo and none of them rated the shampoo as bad on these issues. 76.6% and 85% of the consumers were satisfied with the rinsing and cleansing ability of the shampoo, and no one voted bad on these issues. According to the obtained results, most users were satisfied with the foaming, rinsing, spreading, and detergency ability of formulated herbal shampoo, and consequently, these four items on the questionnaire were evaluated as very good. Gender-based difference in answers was not statically significant \[Tables [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}\]. Most of the users believed their hair became dry and conditioned after using the formulated herbal shampoo. Most users had no hair loss problem. Only 19% of the consumers reported increase in hair loss after usage of this shampoo. Usage of the prepared herbal shampoo showed good results in obviating itch, dandruff, and spout. The majority of users had no sensitivity with their scalp and eyes. 54--60% of the consumers struggling with symptoms embracing itching, dandruff, and spout. The viscosity of the licorice shampoo was better than the commercial and nonherbal shampoo due to the nature of saponins that exist in the *G. glabra* extract; however, consumers were dissatisfied with the odor of shampoo. More specific study is required to determine the effectiveness of the shampoo in reducing hair and scalp irritation. Different studies formulated herbal shampoos; for instance, Aghel *et al*.\[[@ref15]\] formulated a herbal shampoo by employing 5% w/w total saponins of *Acanthophyllum squarrosum*. The shampoo was evaluated for the appearance, foaming capability, and viscosity. The detergency ability was determined to be 67.33% using Thompson test method; furthermore, the formulated shampoo possessed rheological behavior.

Rakesh *et al*.\[[@ref36]\] used a mixture of *Asparagus racemosus, Acacia concinna*, and *Sapindus mukorossi* to formulate a herbal shampoo. Glycerin, EDTA, xanthan gum, Methylparaben, water and orange oil used in the formulation. The pH of the formulation was adjusted to 5.5. Al Badi and Khan\[[@ref31]\] formulated a herbal shampoo using extracts of *Acacia concinna, Sapindus mukorossi, Phyllanthus emblica, Ziziphus spina-christi*, and *Citrus aurantifolia*. The shampoo was clear in visual appearance, and had a pH of 7.02, wetting time of 187 ± 4s, and foam volume of 113 and 115 ml. Furthermore, amount of solid contents, detergency, and so on were tested.\[[@ref31]\] Shinde *et al*.\[[@ref37]\] used the extracts of *Brassica nigra, Cassia fistula, Cassia tora, *Glycyrrhiza glabra*, Saussarea lappa, Bucchania lanzan*, and *Emblica officinalis* in an antidandruff shampoo formulation. The pH of the formulated shampoos varied from 7.31 to 7.75. The foam volume of the preparation was set to be 54--83 ml. The wetting time was regulated to be 11.66--23.66 s through Draves' test. The detergency ability of the prepared shampoos varied from 78.89% to 84.18%.\[[@ref37]\]

C[ONCLUSION]{.smallcaps} {#sec1-6}
========================

In this study, an aqueous extract of licorice was used to formulate a herbal shampoo as it has been used traditionally for hair treatment; in the cosmetic industry, it is used because of the existence of saponins and phytoconstituents. To formulate a product that is compatible with standards, several tests should be executed including physicochemical evaluation and microbial control assessment, and also customers should be polled to know the performance quality and the adverse effects. The product was safe and showed acceptable results in the evaluation tests. The formulated herbal shampoo was beneficial in hair and scalp irritation including itch, dandruff, spout, and so on. Specific clinical investigations are required to establish the efficiency of formulated herbal shampoo on obviation of hair and scalp problems. The present study shows that the shampoo product from licorice has good utility. However, further research is required to prove its quality both in formulation and consumption performance.

Financial support and sponsorship {#sec2-7}
---------------------------------

The authors appreciate Shiraz University of Medical Sciences for their financial support.

Conflicts of interest {#sec2-8}
---------------------

There are no conflicts of interest.
