Development of Antidandruff Shampoo from the Fermented Product of Ocimum Sanctum Linn

cosmetics

Article Development of Antidandruff Shampoo from the Fermented Product of Ocimum sanctum Linn.

Chanun Punyoyai 1, Sasithorn Sirilun 2,3, Panuwan Chantawannakul 4 and Wantida Chaiyana 2,3,* ID

1 Master’s Degree Program in Cosmetic Science, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand; [email protected] 2 Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand; [email protected] 3 Innovation Center for Holistic Health, Nutraceuticals, and Cosmeceuticals, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand 4 Bee Protection Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand; [email protected] * Correspondence: [email protected]; Tel.: +66-53-944-430

Received: 5 June 2018; Accepted: 13 July 2018; Published: 15 July 2018

Abstract: This study aimed to investigate Malassezia furfur inhibitory activity of the fermented product from Ocimum sanctum and develop an antidandruff shampoo. The fermented product was obtained by the fermentation process of the aerial part of O. sanctum. Total soluble protein was detected in the fermented product with the amount of 65.32 ± 0.14 mg/100 mL, whereas there was no organic acid. The inhibitory activity against four strains of M. furfur (No. 133, 656, 6000, and 7966) of the fermented product and shampoos containing the fermented product were investigated by broth dilution and agar diffusion method, respectively. The fermented product possessed high antifungal activity with the minimum inhibitory concentrations for 50% (MIC50) of M. furfur 133, 656, 6000, and 7966 of 0.125, 0.25, 0.125, and 0.125 mg/mL, respectively. Interestingly, the antifungal activity against M. furfur 656 was comparable to that of ketoconazole. Shampoo formulation C, which was the best formulation in terms of characteristics and stability, obtained a high level of satisfaction scores in terms of hair smoothness, hair shine, ease in combing, frizz reduction, and triboelectric reduction while brushing. Additionally, the shampoo containing 2% (w/w) of the fermented product of O. sanctum also possessed inhibitory activity against M. furfur 133, 656, 6000, and 7966 with inhibition zones of 13.2 ± 1.6, 12.8 ± 1.1, 18.7 ± 0.3, and 17.0 ± 1.1 mm respectively. Therefore, this shampoo was suggested for use as an antidandruff shampoo.

Keywords: Ocimum sanctum; Malassezia furfur; antidandruff; shampoo; fermentation

1. Introduction Nowadays, people pay more attention to cleansing themselves than in the past. For this reason, cleansing products are becoming popular, especially shampoo, which is very important in daily life in order to clean the scalp, remove excess oils or dandruff, and eliminate dead cells [1]. However, most of the commercial shampoos that are available in the market could cause scalp problems, such as dandruff, allergies, or irritation, and do the opposite to what consumers want and how shampoos are supposed to perform. Dandruff, considered as a group of skin cells overproduced on the scalp, is the main scalp problem, and afﬂicts more than half of the people in the world [2]. Normally, each skin cell sheds individually, but sometimes, several cells accumulate and this leads to dandruff. The symptoms of dandruff can be

Cosmetics 2018, 5, 43; doi:10.3390/cosmetics5030043 www.mdpi.com/journal/cosmetics Cosmetics 2018, 5, 43 2 of 12 an abnormal epidermis, scalp irritation, itching, and large scalp flakes [3]. Another important factor associated with dandruff is an abundance of Malassezia sp., which affects sensitive skin and causes scalp irritation [4,5]. Most commercial antidandruff shampoos employ chemicals as active ingredients. These chemicals may cause serious hair and scalp problems, such as irritation, itchy eyes, hair damage, and allergies. Therefore, natural compounds, which are friendly to the skin, could help to ease those problems. Consequently, the hair care products consisting of natural ingredients become more popular. People have recently become more focused on the benefits of natural products, particularly herbs. Therefore, natural compounds are widely used as remedial agents. Ocimum sanctum Linn. has been well documented for its therapeutic potential, including usages for the treatment of wounds, catarrhal fever, otalgia, lumbago, hiccough, ophthalmia, gastric disorders, genitourinary disorders, skin diseases, various forms of poisoning, and psychosomatic stress disorders [6]. O. sanctum has also been investigated for various biological activities, including antimicrobial, antifungal, anti-septic, anti-stress, anti-cancer, and antioxidant activity [7–10]. Therefore, O. sanctum could have the potential to be used as a natural antidandruff agent. There are various methods for extracting the bioactive compounds from O. sanctum [11–13]. However, fermentation by Lactobacillus plantarum was selected for the present study. The fermentation process is eco-friendly, and it improves the efficacy and lowers the side effect of medicinal plants. Moreover, the antidandruff activity of the fermented product from O. sanctum has not been reported before. Therefore, the aim of this study was to investigate the inhibitory activity against Malassezia furfur of the fermented product from O. sanctum. Additionally, shampoos containing the fermented product from O. sanctum were developed and characterized.

2. Materials and Methods

2.1. Plant Materials The whole plant of O. sanctum was collected from Chiang Mai, Thailand. The plant was authenticated and voucher specimen number 023230 was deposited in the ofﬁcial Herbarium of the Faculty of Pharmacy, Chiang Mai University, Thailand. The plants were cleaned and dried in the oven at the temperature of 50 ◦C. The dried plants were then ground into powder using an electric blender (Panasonic-1061, Panasonic, Kadoma, Japan).

2.2. Microorganisms Lactobacillus plantarum was obtained from the Health Innovation Institute, Chiang Mai, Thailand. Four strains of M. furfur, including 133, 656, 6000, and 7966, were isolated from clinical samples by the Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.

2.3. Preparation of Extracts % The dried plant powder of O. sanctum was extracted by the previously optimized fermentation process [14]. Before the fermentation, starter culture was prepared by activating the L. plantarum in De Man Rogosa and Sharpe (MRS) broth and incubated at 35 ± 2 ◦C for 24 h. The dried plant materials were mixed with water and cane sugar at the ratio of 0.5:10:1 in a sterile tank and inoculated by 10% w/w of L. plantarum culture (107 CFU/mL). The mixture was then incubated at 35 ± 2 ◦C for 30 days [15]. The fermented plant juice was collected and then ﬁltered through sterile No.1 Whatman ﬁlter paper and dried using a freeze dryer (FreeZone 4.5 model 7750031, Labconco, Kansas, MO, USA). The fermented product was kept at 4 ◦C until further use.

2.4. Determination of Antifungal Susceptibility Testing of the Fermented Product of O. sanctum The antifungal susceptibility test of M. furfur was determined by broth dilution method [16]. Four M. furfur strains, including No. 133, 656, 6000, and 7966, were isolated, sub-cultured onto modiﬁed Cosmetics 2018, 5, 43 3 of 12

Dixon agar (3.6% malt extract, 0.6% peptone, 2% ox bile, 1% Tween 40, 0.2% glycerol, 0.2% oleic acid, 1% olive oil, and 1.5% agar), and incubated at 32 ± 2 ◦C for 3–5 days before the antifungal susceptibility test. The fungal cells were suspended in 1% (w/w) Tween 80 in order to obtain an optical density of 2.4 McFarland, approximately 106 CFU/mL [16]. The sample was dissolved in dimethylsulfoxide (DMSO) using various concentrations, ranging from 0.004 to 1 mg/mL. Ketoconazole dissolved in DMSO at a concentration of 0.5 mg/mL was used as a positive control. Then, 100 µL of each ﬁltrated sample was mixed with 100 µL of microbial suspension (106 CFU/mL) and incubated at 32 ± 2 ◦C for 48 h. The absorbance of each mixture was then measured at the wavelength of 565 nm by a microplate reader (Biochrom, Cambridge, UK). The results were reported as being minimum inhibitory concentrations for 50% (MIC50), which was the concentration that resulted in 50% growth reduction when compared with the extract-free SDB. The minimal fungal concentrations (MFCs) assay was performed after the MICs method. The sample of the previous MICs assay was streaked on the modiﬁed Dixon agar. After the incubation at 32 ± 2 ◦C for 72 h, MFCs was determined as the lowest concentration, which showed no microbial growth. The experiments were performed in triplicate.

2.5. Determination of Organic Acids Content by High Performance Liquid Chromatography (HPLC) The fermented product was analyzed for its organic acids content, including lactic acid and acetic acid, using HPLC analysis. The fermented product was dissolved in DMSO at the concentration of 1 mg/mL and filtered through a 0.45 µm HPLC filter (Millipore SLCR013NL, Millipore, Bedford, MA, USA). The filtered solution was further assessed by HPLC with an auto-sampler (Model HP 1100, Agilent Tecnologies, CA, USA). The injection volume was 20 µL. Separation was performed on a reversed phase column, which was the Phenomenex Luna® C18 (2) (150 mm × 4.6 mm, 5 µm) with a guard column (Phenomenex C18, 4 mm × 3 mm, 5 µm). The mobile phase, which was a 0.1% (v/v) phosphoric acid solution (pH 5.0), was allowed to flow at the rate of 0.5 mL min−1 at the temperature of 25 ◦C. The HPLC analyses were performed with a UV detector set at 210 nm.

2.6. Determination of Total Soluble Protein Content Total soluble protein content of the fermented product was determined by the Lowry’s method [17]. Brieﬂy, 5 mL of the extract solution (1 mg/mL) was mixed with 50 mL of phosphate buffer (pH 7.0 ± 0.2). Then, 2 mL taken from the above solution was mixed with 2 mL of 20% trichloroacetic acid. After incubation at room temperature for 30 min, the mixture was centrifuged at 3000 rpm for 25 min, washed with acetone twice, and centrifuged again. The supernatant was then removed and the precipitate was dissolved in 5 mL of 0.1N NaOH solution. After that, 1 mL of the solution was mixed with 5 mL alkaline copper sulphate reagent and incubated at room temperature for 1 min. Then, 0.5 mL Folin’s reagent was added and allowed to stand for 30 min. The absorbance of the ﬁnal solution was measured by using a UV spectrophotometer (Shimadzu UV-2450, Shimadzu, Kyoto, Japan) at 660 nm. Bovine serum albumin (BSA) was used as a standard compound.

2.7. Development of Shampoo Formulations The shampoos were prepared using various ingredients. Different types of detergent, including anionic, sodium lauryl ether sulfate, and ammonium lauryl sulfate, were used as major ingredients of the shampoo formulations. Additional additives, such as preservative, foam builder, emollient, humectant, thickening agent, pearlescent, conditioning agent, antioxidant, pH modiﬁer, solubilizing agent, and opacifying agent, were also added. The ingredients of each shampoo formulation are shown in Table1. Cosmetics 2018, 5, 43 4 of 12

Table 1. Ingredients of shampoo formulations in this study.

Formulation (% w/w) Ingredient Function ABCDEFGH Sodium laureth sulfate (70%) Detergent - 12 20 12 12 15 8 15 (Texapon N70) Sodium laureth sulfate (28%) Detergent 10 - 5 - - - 10 5 (Texapon N8000) Cocamide diethanolamine Foam builder, 15 5 5 5 5 10 12 3 (Com KD) thickening agent Cetrimonium chloride (CT 429) Detergent 5 3 3 3 3 3 3 3 Cocamidopropyl betaine (55AB) Detergent 8 10 8 8 8 8 7 5 Cetyl alcohol Emollient, opacifying ------3 - agent Propylene glycol Humectant 1.5 - 1 1 1 1 1.5 1 Glycerin Humectant - 2 ------Lanolin Emollient 0.5 ------2 Sodium lauryl ether sulfate cocamide Pearlescent - - 10 8 7 10 5 - meapearl shine concentrate Sodium chloride (conc. 0.5 mg/mL) Thickening agent 4 - - - - 2.5 - 1 Tween 80 Solubilizing agent - - 1 0.5 0.5 1 - 2 Rice bran oil Emollient 2 ------Mineral oil Emollient ------1 1 Wheat protein Conditioning agent - - - - 1 - 1 - Vitamin E Antioxidant - - - - - 1.5 - - Citric acid Preservative, pH q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s. modiﬁer Water q.s. Diluent 100 100 100 100 100 100 100 100

2.8. Characterization of Shampoo Formulations Shampoos formulated in the present study were characterized in a comparison with the commercial shampoos and Texapon N70. The codes of each shampoo investigated in the present study are shown in Table2.

Table 2. Codes of shampoo samples.

Shampoo Formulation Code Shampoo base A, B, C, D, E, F, G, H Shampoo containing fermented product FO2, FO5, FO10 Texapon N70 N70 Nizoral C1 Head and shoulder C2 Clear C3 Kokliang C4

2.8.1. Physical Appearance Approximately ﬁve grams of each sample was evaluated for the physical appearance by organoleptic inspections in terms of external appearance, homogeneity, color, and odor.

2.8.2. Determination of Foaming Ability and Foam Stability Foaming ability was determined using the cylinder shake method [18]. Briefly, 1 g of sample or commercial shampoo was added into a cylinder and 50 mL of DI water was then added. The cylinders were covered with paraffin film and shaken ten times. The foam volume that was recorded immediately was reported as flash foam and the foam volume recorded four minutes after the shaking was reported as maximum foam. The experiment was performed in triplicate. Cosmetics 2018, 5, 43 5 of 12

2.8.3. pH Measurement The formulation was diluted in DI water in order to obtain the ﬁnal concentration of 10% (v/v). The pH was measured by using a pH meter at room temperature. The experiment was performed in triplicate.

2.8.4. Determination of Wetting Time The wetting time of the shampoo was investigated [19]. A paper was cut into a one-inch square and placed on the aqueous solution of 1% (w/w) shampoo. The time when the paper started becoming wet was recorded and reported as the wetting time. The experiment was performed in triplicate.

2.8.5. Determination of Solid Content The solid content of the shampoo was investigated [18]. First, 4 g of the sample was placed in an evaporating dish and the total weight was recorded. The evaporating dish was placed in a water bath to allow evaporation to occur until it was completely evaporated. Then, the evaporating dish was weighed again. The percentage of solid content was then calculated using the follow equation:

% solid content = [((A − B)/4) × 100] (1) where A was the total weight of the sample and evaporating dish after evaporation and B was the total weight of the sample and evaporating dish before evaporation. The experiment was performed in triplicate.

2.8.6. Viscosity Measurement The viscosity of each formulation was determined by using a Brookﬁeld Rheometer (Model R/S-CPS, Brookﬁeld Engineering Laboratories, Inc., Middleboro, MA, USA) at room temperature. The experiment was performed in triplicate.

2.9. Stability Test of Shampoo Formulations The stability tests of the shampoo formulations were determined by a heating–cooling cycle. The formulations were kept in a refrigerator at 4 ◦C for 24 h and in a hot air oven at 45 ◦C for 24 h. This heating–cooling cycle was repeated eight times. After that, the formulations were characterized in terms of physical appearance, pH, viscosity, solid content, foaming ability, and wetting time.

2.10. Evaluation of Conditioning Performance of Shampoo Formulations The conditioning performance of the shampoo formulations was determined with slight modiﬁcations [20]. A tress of hair was washed by shaking it in a mixture of 10 g of the sample and 30 g DI water for 2 min in an Erlenmeyer ﬂask. The tress of hair was then removed and rinsed with DI water until clean. Each tress was kept at room temperature until it was completely dry. The conditioning effects of the shampoo were evaluated by questionnaires in terms of smoothness, hair shine, ease in combing, frizz reduction, and triboelectric reduction. The questionnaires were answered by thirty volunteers after the examination of the tresses of hair. The conditioning effects were rated from 1 to 5 (1 = very poor; 2 = poor; 3= moderate; 4 = good; 5 = excellent).

2.11. Determination of Antifungal Susceptibility Testing of Shampoo Containing the Fermented Product of O. sanctum by Agar Well Diffusion Method Four M. Furfur strains, including No. 133, 656, 6000, and 7966, were isolated, sub-cultured onto modiﬁed Dixon agar and incubated at 32 ± 2 ◦C for 3–5 days before the antifungal susceptibility test. The fungal cells were suspended in 1% Tween 80 in order to obtain an optical density of 2.4 McFarland, approximately 106 CFU/mL. Cosmetics 2018, 5, 43 6 of 12

M. furfur was spread on the modiﬁed Dixon agar. The plates were holed with a sterile cork borer and 100 µL of each sample was added into them. The culture plates were incubated at 32 ± 2 ◦C for 48 h and the diameter (mm) of the zones of inhibition were measured. A formulated shampoo was used as a negative control. Commercial anti-dandruff shampoos (Nizoral and Head and Shoulder) were used as positive controls. The experiments were performed in triplicate.

2.12. Statistic Analysis Statistical significance was assessed by the one-way analysis of variance (ANOVA) using the SPSS 17.0 for Windows (SPSS Inc., Chicago, IL, USA). The level of significant difference was defined at p < 0.05.

3. Results and Discussion

3.1. Yield of the Fermented Product of O. sanctum The physical appearance of the fermented product of O. sanctum was a brown semisolid mass with an agreeable odor. The yield was 11.93% (w/w). The fermented product of O. sanctum might contain phenolic compounds, sugars, organic acids, and pigments, which were released from the plant cell during the fermentation process [21].

3.2. Antifungal Susceptibility Testing of the Fermented Product of O. sanctum Antifungal activities against four strains of M. furfur are presented in Tables3 and4. The fermented product of O. sanctum was a potent extract that could inhibit all strains of M. furfur. Interestingly, the fermented product of O. sanctum possessed the comparable antifungal activity against M. furfur No. 656 to that of ketoconazole, a well-known broad-spectrum antifungal drug. The results were in agreement with the previous study, which reported that the fermented product could enhance the antibacterial activities of Rheum palmatum L. when compared with a non-fermented product [22]. Besides, several microbes, which were used as intermediate producers in the fermentation process, could break down the plant cell membrane and allow the bioactive compounds to release from the cells easier [23]. Moreover, some intermediate microbes could change the herbal ingredients to increase inhibitory activity [23]. Therefore, the fermented product could be used in various applications because of its inhibitory activity against M. furfur, which is known as the cause of several chronic skin diseases, such as dandruff, seborrhoeic dermatitis, pityriasis versicolor, atopic dermatitis, psoriasis, and both conﬂuent and reticulate papillomatosis [24].

Table 3. The minimum inhibitory concentrations required for 50% growth reduction when compared

with the extract-free Sabouraud Dextrose Broth (MIC50) and minimal fungal concentrations (MFCs) of the fermented product of O. sanctum against four strains of M. furfur.

M. furfur 133 M. furfur 656 M. furfur 6000 M. furfur 7966 Sample MIC50 MFCs MIC50 MFCs MIC50 MFCs MIC50 MFCs (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) Ketoconazole 0.0078 0.0078 0.25 0.25 0.0625 0.0625 0.125 0.125 Fermented product 0.125 0.125 0.25 0.25 0.125 0.5 0.125 0.5 CosmeticsCosmeticsCosmetics Cosmetics2018 2018,Cosmetics 20185,, x5 ,FOR2018, x5 ,FOR x , PEER 2018FOR5, PEERx FOR, PEER 5REVIEW, x REVIEW PEERFOR REVIEW PEER REVIEW REVIEW 7 of7 12of7 12of 712 of 127 of 12 CosmeticsCosmeticsCosmetics Cosmetics2018 2018, Cosmetics 20185,, x5 ,FOR2018, x5 ,FOR x, 2018PEER FOR5, PEERx ,FOR PEER5 REVIEW, x REVIEW FORPEER REVIEW PEER REVIEW REVIEW 7 of7 of127 12of 127 of 127 of 12

Cosmetics 2018, 5, 43 7 of 12

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Development ofEight ofthe theof Shampoo theformulations Shampooof Shampoothe of Shampoo theFormulation Formulation Shampoo Formulation of Formulation different Formulation shampoo types were developed, including creamy, clear, EightEightEight formulations Eightformulations formulationsEight formulations formulations of ofdifferen ofdifferen differenof different ofshampoot shampoodifferent shampoot shampoo typest shampootypes types were typeswere were typesdeveloped weredeveloped developed were developed, developedincluding, including, including, including creamy,, includingcreamy, creamy, creamy, clear, clear, creamy, clear, and clear,and and clear, and and and pearlescent shampoo. The creamy shampoos include formulations A and H; the clear shampoo pearlescentpearlescentpearlescentEightEightpearlescentEight formulations pearlescent shampoo.Eightformulations shampoo.formulations shampoo.Eight formulations shampoo. formulationsThe shampoo. ofThe ofThedifferencreamy of differen creamyThe differencreamyof The differentcreamy shampoosofshampoo t shampoos shampoodifferen tcreamy shampoosshampoo tshampoos shampoo types tinclude shampoos shampootypesinclude typesinclude were types includewereformulation were formulation typesdeveloped include formulationdevelopedwere developedformulation were developedformulations ,As developedincluding , A sandincluding , A andincludings and,H;A including H;s and thecreamy,,AH; includingthecreamy, and clearH;thecreamy, clear thecreamy, H;clear clear,shampoo clear, cleartheshampoocreamy, clear,shampoo andclear clear,andshampoo and clear, shampoo and and includes formulation B; and the pearlescent shampoos include formulations C, D, E, F, and G. Based on includespearlescentpearlescentincludespearlescentincludesincludespearlescent formulation formulation pearlescentincludesshampoo. formulation shampoo. shampoo.formulation shampoo. formulation B; The B;andshampoo. The B;and The creamyandthe B;creamy theThe andpearlescentcreamy the B; pearlescent The creamy andtheshampoospearlescent shampoos creamypearlescenttheshampoos shampoospearlescent shampoos include shampoosshampoos include shampoosinclude include includeshampoosformulation include formulation include includeformulation includeformulation formulation formulation include formulationformulations A sformulation Asand Aformulationands s C, andsH;A C,D,sH; s andC, theD, H;AE, s theD, C,E, F,andcleartheH; E, F,sD,andclear C, F,theand clearH;E, shampooD,andG. F,the shampooclear G.E, Basedand shampooG. BasedF,clear BasedshampooandG. Based shampooG. Based the physical appearance inspection, shampoo formulations A, B, C, G, and H had homogeneous onincludesincludeson theincludeson the physicaltheonincludes formulationphysical theformulationphysicalincludeson formulation physicalthe appearance formulation appearance physical appearance formulation B; B;appearanceand B;and appearance inspectionandthe B; inspectionthe and pearlescentinspectionthe B; pearlescent inspectionandthepearlescent, shampoo inspectionpearlescentthe, shampoo, shampoopearlescentshampoos shampoos, shampoo shampoos formulation, shampooformulationshampoos formulation include shampoos includeformulation include formulations includeformulationA,s formulationA, s B, include formulationA, B,C,s B,formulationA, C,G, C, sG, B, andformulationsA, G,C,ands B, C, andHsD,G, C, HD,had E,and s HD,G,had E,C,F, hadhomogeneous E, andF,HsandD, homogeneousC, F,andhad E, homogeneous HD,andG. F, G.had homogeneousE,Based and G. BasedF, homogeneous Based andG. Based G. Based textures. Meanwhile, formulations D, E, and F were separated into two layers. Therefore, these unstable textureonontexture theontexture the sphysicalthetextureon. physicalsMeanwhile,. thephysicalsMeanwhile,ontexture. Meanwhile, sphysicalthe .appearance Meanwhile, appearance physicals .appearance Meanwhile,formulation appearanceformulation formulation appearanceinspection formulationinspection inspection formulations inspectionD,s D,, s E, shampooinspection,D, shampooE,sand, E,shampooD,and s ,and FE,shampooD, Fwereformulationand, E,shampooformulationFwere formulationwere and Fseparated wereformulationseparated Fseparated formulationweres A,separateds A, sB,into A,separated B,intoC,s B,intoC, A,twoG, C, twosG, intoB, andA, twoG, layers.andC, B,intolayers. and twoH G, layers.C, Hhad and twoHG,hadTherefore,layers. hadTherefore,homogeneous andH Therefore,layers.homogeneous had homogeneous HTherefore, hadhomogeneous theseTherefore, these homogeneous these these these formulations were excluded from the further studies. Only formulations A, B, C, G, and H were unstabletexturetextureunstabletextureunstablesunstabletexture. formulationssMeanwhile,. formulationssMeanwhile,textureunstable. formulationsMeanwhile,s. formulationsMeanwhile,s. formulationsMeanwhile, formulationwere formulationwere formulationwere excluded formulationwereexcluded excluded formulationswere excludedD,s fromD,s E,excludedfromD, E,sfromand the E,D,and thefrom s and furtherFE,theD, Ffurtherwerefromand theE,furtherFwere were and studies.furtherFtheseparated studies.wereseparated furtherFstudies.separated were studies.Onlyseparated Only into studies. separatedOnly intoformula intoOnlyformulatwo formulatwointo Onlytwo layers.formulation layers. intotwotion layers.formulastion A, stwoTherefore,layers. A,tion sB,Therefore, A, B,Therefore,layers.C,stion B, C,A, G,Therefore, C, sG, B,and A, these G,Therefore,C,and theseB, andH G,these C, H and HG,these and Hthese H selected for the future characterizations. Foaming ability, which is a crucial property of shampoo to wereunstableunstablewereunstablewere selected wereunstableselected formulations selected formulationsunstablewere formulationsselected for formulationsforselected thefor formulationsthe wereforfuturethe werefuture werethefor futureexcluded excludedwerecharacterizationsthefuture excludedcharacterizations werecharacterizationsfuture excluded fromcharacterizations fromexcluded fromcharacterizations the thefrom furtherthe. furtherFoamingfrom. thefurtherFoaming. Foaming studies.furtherthe. studies.Foaming furtherstudies.ability,. Foamingability, studies. Onlyability, Only studies. ability,Onlywhich formulawhich formula Onlyability,which formula isOnlywhich formulationisa tioniswhicha crformulas tion ucialacrA,iss ucial crA,tion sB,a ucialA, is B,crpropertyC, stion B, aucialC, propertyA,G, cr C,property sG, B, anducialA, G, andpropertyC, B, andof HG, C,property Hof and ofHG, andofH ofH the consumer, is considered as an important parameter in the evaluation of shampoo. The result of werewere were selected wereselected selectedwere selected for forselected thefor the forthefuture future forthefuture characterizationsthefuture characterizations characterizationsfuture characterizations characterizations. Foaming. Foaming. Foaming. Foaming ability,. Foamingability, ability, ability,which which ability,which iswhich isa iswhichacr ucialacris ucialcr a ucial iscrproperty aucialproperty crpropertyucial property of property of of of of foaming ability in Table5 showed that the ﬂash foam and maximum foam of shampoo formulations B, C, and H were similar to that of the commercial products, whereas formulation A could only produce a small amount of foam.

Table 5. Characterization of formulated and marketed shampoo.

Maximum Wetting Sample Flash Foam pH % Solid Viscosity Foam Time (min) A 16.67 ± 0.58 20.67 ± 0.58 7.10 ± 0.02 25.78 ± 0.48 15.81 ± 0.55 0.26 ± 0.03 B 46.67 ± 2.89 62.67 ± 4.62 6.17 ± 0.01 21.02 ± 0.05 32.80 ± 0.55 0.66 ± 0.01 C 46.67 ± 1.53 68.33 ± 2.89 7.81 ± 0.02 28.55 ± 0.37 26.58 ± 0.98 1.18 ± 0.03 G 32.33 ± 2.52 48.00 ± 2.65 8.58 ± 0.02 35.11 ± 0.13 22.52 ± 1.85 1.28 ± 0.07 H 44.67 ± 1.53 56.67 ± 1.53 7.32 ± 0.01 28.17 ± 0.33 18.48 ± 1.54 2.32 ± 0.03 C1 44.00 ± 1.73 59.33 ± 1.15 7.39 ± 0.01 23.41 ± 0.09 21.80 ± 1.94 3.91 ± 0.11 C2 47.33 ± 2.08 65.00 ± 3.00 7.64 ± 0.01 23.79 ± 0.02 32.95 ± 0.83 0.83 ± 0.02 C3 48.00 ± 2.00 70.67 ± 1.15 7.41 ± 0.01 22.98 ± 0.07 37.44 ± 1.24 1.42 ± 0.04 C4 45.67 ± 2.08 59.00 ± 3.61 7.48 ± 0.01 19.43 ± 0.02 24.18 ± 0.68 1.22 ± 0.02 Cosmetics 2018, 5, 43 8 of 12

The pH of each shampoo is shown in Table5. The pH of commercial shampoos (C1, C2, C3, and C4) were in the range of 6 to 7.5, which were neutral and good to use on the hair and scalp. Shampoo formulations A, C, and H showed comparable pH to that of the commercial products, whereas shampoo formulation B was a little bit acidic, which would be good for people with healthy hair and scalps because the normal pH of the scalp was 5.5. Moreover, the mild acidity of the shampoo could increase the hair’s quality, decrease eye irritation, and maintain the ecological balance of the scalp [26]. However, shampoo formulation G had higher pH than that of the commercial products, which would not be good for usage because the high pH would decrease the performance of the product concerning compatibility, irritation, friction, and frizz effect [27]. The solid content of each shampoo is shown in Table5. Formulations A, B, C, and H had solid contents in the normal range of 20% to 30%, whereas formulation G had a higher solid content (35%). Commercial products, including C1, C2, and C3 had solid contents in the range of 20% to 30%, whereas C4 had a solid content lower than 20%. In general, the appropriate solid content for shampoo is between 20% and 30% [18]. Consequently, the shampoo with lower solid content could be rinsed out easily. The wetting times of all the developed formulations were similar to that of the commercial products (Table5). Since the wetting time relies on the wetting ability of the shampoo and normally depends on the concentration of the detergents in the formulation, the shampoos with high wetting times would be good for cleaning because they could diffuse and wet the hair shaft very well [19]. The viscosity of each shampoo is shown in Table5. It was noted that the viscosity of the commercial shampoos varied. C1 had a very high viscosity. Most of the commercial shampoos had a viscosity in the range of 0.8 Pas to 1.4 Pas. Shampoo formulations B, C, and G had a similar viscosity to most of the commercial shampoos.

3.5. Evaluation of Conditioning Performance of Shampoo Formulation The tresses of hair were evaluated for conditioning performance of the shampoo formulations by thirty volunteers, the majority of which were female (86.7%) aged between 21 and 30 years old. Shampoo formulations A, B, C, G, and H, which showed good characteristics and good stability, were evaluated for satisfaction compared to N70 and commercial antidandruff shampoos, including C1 and C2. The conditioning performances of each shampoo are shown in Table6. The results noted that volunteers were more satisﬁed by shampoo formulations A, B, C, G, and H than N70. The satisfaction scores of the developed shampoos were compared to that of the commercial antidandruff products in terms of smoothness, hair shine, ease in combing, frizz reduction, and triboelectric reduction. Interestingly, formulation C got the highest satisfaction score for smoothness (3.8 ± 0.8), hair shine (3.8 ± 0.8), ease in combing (3.6 ± 0.8), frizz reduction (3.4 ± 0.9), and triboelectric reduction (3.7 ± 1.0).

Table 6. Satisfaction scores of shampoo formulations in the terms of smoothness, hair shine, ease in combing, frizz reduction, and triboelectric reduction.

Shampoo formulation Parameter A B C G H N70 C1 C2 Smoothness 3.3 ± 0.9 3.8 ± 0.8 3.8 ± 0.7 3.6 ± 0.6 3.4 ± 0.8 2.6 ± 0.8 3.7 ± 0.9 3.5 ± 0.9 Hair shine 3.1 ± 0.8 3.3 ± 0.7 3.8 ± 0.8 3.5 ± 0.6 3.5 ± 0.8 2.9 ± 0.6 3.5 ± 0.8 3.6 ± 0.7 Ease in combing 3.1 ± 0.8 3.2 ± 0.7 3.4 ± 0.9 3.3 ± 0.7 3.4 ± 0.8 2.4 ± 1.0 3.1 ± 1.0 3.5 ± 1.0 Frizz reduction 3.2 ± 0.7 3.0 ± 0.8 3.6 ± 0.7 3.8 ± 0.6 3.3 ± 0.6 2.4 ± 0.8 3.2 ± 0.9 3.6 ± 0.7 Tribolectric reduction 3.1 ± 0.9 3.3 ± 0.9 3.7 ± 0.9 3.5 ± 0.7 3.3 ± 0.9 2.4 ± 0.8 3.0 ± 0.9 3.1 ± 1.0

The best formulation that got the highest overall satisfaction score was shampoo formulation C, which was a creamy shampoo with the ﬂash foam and maximum foam of 46.67 and 68.33 mL, respectively. The pH was 7.81, which is an appropriate value for use on the scalp. The solid content was Cosmetics 2018 , 5, x FOR PEER REVIEW 9 of 11

activity againstCosmetics 2018 M., 5furfur, 43 133, M. furfur 6000, and M. furfur 7966 with the inhibition zones9 ofof 12 7.3 ± 0.6, 16.0 ± 1.0, and 14.6 ± 0.8 mm, respectively. Moreover, FO2, FO5, and FO10 had less antifungal activity against the four strains of M. furfur than the C1 and C2. 28.55%, which is perfect for rinsing out. The wetting time was 26.58 min and the viscosity was 1.18 Pas. ShampoosFurthermore, containing this formulation different got concentrations the highest satisfaction of the scorefermente in termsd product of smoothness, of O. hairsanctum shine, did not show differentease in combing,antifungal frizz activities reduction, ( andp > 0.05). triboelectric However, reduction. FO10 Therefore, was not shampoo stable formulationas it separat C wased into two layers afterselected the forheating–cooling the incorporation stability of the fermented test. Th producterefore, from FO2O. was sanctum. suggested for further use because the higher amount of the extract could not enhance the M. furfur inhibitory activity. Additionally, the 3.6. Antifungal Susceptibility Testing of Shampoo Containing the Fermented Product of O. sanctum inhibitory effect of the shampoo formulations could be as a result of other ingredients that also w w possess antimicrobialThe results of antifungal activity, activity such of as the shampoos sodium containing laureth 2% sulfate, (FO2), 5% cocamide (FO5), and 10% diethanolamine, ( / ) (FO10) of the fermented product of O. sanctum against M. furfur 133, M. furfur 656, M. furfur 6000, cocamidopropyland M. furfur betaine,7966, respectively, cetrimonium compared chloride, with commercial and p antidandruffropylene shampooglycol [28–31], and formulation because C these compounds,are shown especially in Table 7 cationicand Figure 1 surfactants,. The results noted could that i thenteract FO10 had with a larger the inhibition cellular zone membranes than of microorganismsthose of the [32]. shampoos The containing use of FO FO2 could and FO5 be of combine the fermented with product the otherof O. sanctum ingredients. The inhibition of shampoo, possessingzones antimicrobial against M. furfur activity,133, M. in furfur order656, toM. impr furfurove6000, the and efficacyM. furfur of7966 the were product. 16.1 ± 1.5, 14.6 ± 1.4, 17.2 ± 0.5, and 16.2 ± 1.3 mm, respectively. Meanwhile, formulation C also showed the inhibitory activity against M. furfur 133, M. furfur 6000, and M. furfur 7966 with the inhibition zones of 7.3 ± 0.6, Table 7. Antifungal activity of shampoo containing the fermented product of O. sanctum. 16.0 ± 1.0, and 14.6 ± 0.8 mm, respectively. Moreover, FO2, FO5, and FO10 had less antifungal activity against the four strains of M. furfur than the C1Inhibition and C2. Zone (mm) Sample Table 7. AntifungalM. furfur activity 133 of shampooM. furfur containing 656 the fermentedM. furfur product 6000 of O. sanctum.M. furfur 7966 C 7.3 ± 0.6 7.0 ± 0.0 16.0 ± 1.0 14.6 ± 0.8 Inhibition Zone (mm) C1 Sample 34.5 ± 1.4 37.7 ± 1.4 31.3 ± 1.3 34.3 ± 1.3 C2 38.2M. ± furfur0.5 133 M. 39.4 furfur ± 0.5656 M. furfur 33.16000 ± 0.9M. furfur 7966 37.6 ± 1.9 FO2 C 13.2 ± 7.31.6± a 0.6 12.8 7.0 ±± 0.01.1 a 16.0 18.7± 1.0 ± 0.3 a 14.6 ± 0.8 17.0 ± 1.1 a C1 34.5 ± 1.4 37.7 ± 1.4 31.3 ± 1.3 34.3 ± 1.3 FO5 C2 12.1 ± 38.20.3± a 0.5 12.4 39.4 ± 0.50.3 a 33.1 16.7± 0.9 ± 2.3 a 37.6 ± 1.9 15.0 ± 1.6 a a a a a FO10 FO2 16.1 ± 13.2 1.5± b1.6 14.612.8 ± ± 1.11.4 b 18.7 17.2± 0.3 ± 0.5 a 17.0 ± 1.1 16.2 ± 1.3 a FO5 12.1 ± 0.3 a 12.4 ± 0.3 a 16.7 ± 2.3 a 15.0 ± 1.6 a * The results are FO10expressed as16.1 the± mean1.5 b ± standard14.6 ± 1.4 deviation.b 17.2 The± 0.5 inhibitiona 16.2 zone± 1.3 ofa the cork borer was 7.0 mm.* The resultsFO2, areFO5, expressed FO10 as were the mean shampoos± standard containing deviation. The the inhibition fermented zone of product the cork borer of wasO. sanctum 7.0 mm. at the FO2, FO5, FO10 were shampoos containing the fermented product of O. sanctuma atb the concentration of 2%, 5%, concentrationand 10% of (w 2%,/w), respectively.5%, and 10% The letters( w/wa),and respectively.b denote signiﬁcant The differencesletters and between denote the shampoo significant formulations differences between (thep < 0.05). shampoo formulations ( p < 0.05).

(a) (b)

Figure 1.Figure (a) Antimicrobial1. (a) Antimicrobial susceptibility susceptibility of of shampoos shampoos containing containi the fermentedng the productfermented of O. sanctumproduct. of O. Shampoo containing the fermented product of O. sanctum. Top left: shampoo containing 2% (w/w) of sanctum . theShampoo fermented containing product of O.the sanctum fermented(FO2); topproduct right: shampooof O. sanctum containing. Top 5% left: (w/ wshampoo) of the fermented containing 2% (w/w) of productthe fermented of O. sanctum product(FO5); of bottom O. sanctum left: shampoo (FO2); containing top right: 10% shampoo (w/w) of the containing fermented product 5% (w of/w) of the fermentedO. sanctumproduct(FO10); of O. bottomsanctum right: (FO5); shampoo bottom base left: (Base). shampoo (b) Antimicrobial containing susceptibility 10% (w/ ofw) commercial of the fermented product ofshampoos; O. sanctum top left: (FO10); C1; top bottom right: C2; right: bottom shampoo left: formulation base (Base). C; bottom (b) right: Antimicrobial DI water. susceptibility of commercial shampoos; top left: C1; top right: C2; bottom left: formulation C; bottom right: DI water.

Cosmetics 2018, 5, 43 10 of 12

Shampoos containing different concentrations of the fermented product of O. sanctum did not show different antifungal activities (p > 0.05). However, FO10 was not stable as it separated into two layers after the heating–cooling stability test. Therefore, FO2 was suggested for further use because the higher amount of the extract could not enhance the M. furfur inhibitory activity. Additionally, the inhibitory effect of the shampoo formulations could be as a result of other ingredients that also possess antimicrobial activity, such as sodium laureth sulfate, cocamide diethanolamine, cocamidopropyl betaine, cetrimonium chloride, and propylene glycol [28–31], because these compounds, especially cationic surfactants, could interact with the cellular membranes of microorganisms [32]. The use of FO could be combine with the other ingredients of shampoo, possessing antimicrobial activity, in order to improve the efﬁcacy of the product.

4. Conclusions The fermented product of O. sanctum possessed potent antifungal activity against four strains of M. furfur. Therefore, it could be used as an active ingredient in the development of antidandruff shampoo. Shampoo formulation C was the best formulation because of its good characteristics, good stability, and the fact that it obtained the highest satisfaction score when evaluated by 30 volunteers, especially in terms of smoothness, hair shine, ease in combing, frizz reduction, and triboelectric reduction. Additionally, 2% (w/w) of the fermented product of O. sanctum was suggested for use as an active component in the shampoo as it could inhibit the growth of M. furfur and provide the antidandruff shampoo with a good external appearance and good stability. Additionally, the fermentation process could lower the cost of production, and it is friendly to the environment, harmless, less toxic, and produces less hazardous solvent waste when compared with other extraction methods.

Author Contributions: Conceptualization, W.C.; Methodology, C.P., S.S., and P.C.; Investigation, C.P.; Writing—Original Draft Preparation, C.P. and W.C.; Supervision, W.C., S.S., and P.C.; Project Administration, W.C.; Funding Acquisition, W.C. Funding: This research was funded by Graduated School of Chiang Mai University and Faculty of Pharmacy, Chiang Mai University. Acknowledgments: We would like to thank Mr. Michael Mayo for improving the use of English in the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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