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1 Journal of Biotechnology and Biosafety "#$%&'()*++%',)-./%.0123'40%.01(5,6)(,29, *778(9((2565: Journal of B iotechnology and B iosafety Research article REVIEW ON HAIR, DANDRUFF, SHAMPOO AND EXPERIMENTAL, ANTIMICROBIAL ACTIVITY OF PIROCTONE OLAMINE WHICH IS USED AS ACTIVE IN FORMULATION ANTI DANDRUFF SHAMPOO Nitesh Rajput 1 Vishal Chinchamalatpure 2 * 1 Senoir Manager,R & D, Vitro Med Health Care, Jaipur. 2 Executive, R & D Cosmetic, Sri Sri Ayurveda Trust, Bangalore *Corresponding author id: vishalchinchamalatpure@gmail.com ABSTRACT: In recent days, so many people are facing problems of dandruff. Dandruff causes lots of problems such as itching, scaling, irritation etc. Due to dandruff hair fall takes place giving dull and diminish look to hair. It is reported that Dandruff and hairfall is because of anti-fungal, anti-bacterial property. Hence the present work is a humble attempt to evaluate the definition of hair dandruff Melassezia furfur, Pityrosporum ovale, and candida albicans, which are responsible for the formation of dandruff and shampoo. The quality control of piroctone olamine is of paramount importance for justifying their quality and integrity for acceptance in modern system of cosmeceuticals. Hence the procured sample of the chemical material was first evaluated for their quality by determining colour, odour, ph, solubility, melting point, residue on ignition and % purity. The values obtained within the standard values and hence it was concluded that the procured material was pure to be used for the further experimentation. Key words: hair, dandruff, shampoo, Melassezia furfur, Pityrosporum ovale,, candida albicans, piroctone olamine REVIEW ON HAIR, DANDRUFF, SHAMPOO INTRODUCTION: Hair Hairs, or pili, are present on most skin surfaces except the palm, palmer surfaces of the feet. In adults, hair usually is most heavily distributed across the scalp, in the eyebrows, in the axillae (armpits), and around the external genitalia. Genetic and hormonal influences largely determine the thickness and the pattern of the hair distribution. (Tortora G. J., Derrickson B. H, 2009, p-155) The functions of hair (Phate R. P, 1994, p-244) Protection of scalp from the external damage. To give attractive look and shape to the face and to enhance the facial expression. Healthy hairs enhance beauty. Protect the skin against mechanical trauma and heat loss or high temperature. Protect the scalp against UV radiation. Anatomy of hair (Tortora G. J., Derrickson B. H, 2009) Each hair is composed of columns of dead, keratinized epidermal cells bonded together by extracellular proteins. The shaft is the superficial portion of the hair, which projects above the surface of the skin. The root is the portion of the hair deep to the shaft that penetrates into the dermis, and sometimes into the subcutaneous layer of the skin.

2 Journal of Biotechnology and Biosafety "#$%&'()*++%',)-./%.0123'40%.01(5,6)(,29, *778(9((2565: Journal of B iotechnology and B iosafety A hair is a specialized outgrowth of part of the skin called the epidermis. Outermost lines from the inner root sheath and inner cell from the further hair shaft i.e. the part of hair emerging from the skin is called hair shaft and part which is in the skin is hair root. The epidermis invigilated into the dermis to form a tubal as pocket called hair follicle. The sebaceous gland open directly into the upper part of the follicle, this gland secretes sebum which lubricate and protects the scalp and hair. A smooth muscle called arrector pili muscle which is under autonomic control is connected near the center of hair follicle and runs at an angle towards the epidermis. The center of hair bulb is invigilated to form the dermal papilla for generation and for growth of hair. The cells of hair in contact with dermal papilla are called hair matrix and are responsible for actual production of hair Hair has two distinct parts, the hair follicle and the hair shaft. Hair follicle (Phate R. P, 1994,p-246) Each hair on human body grows form a hair follicle, a tiny saclike hole in human skin. At the bottom of the each follicle is a cluster of special cells that produce to make new hair cells. The new cells that are produced are added on at the root of the hair causing the hair to grow longer. The active hair follicle is divided into 3-segment. Upper segment consist of infundibulum, middle isthmus lies between the duct of the sebaceous gland and the attachment of arrector pili muscles. Lower inferior segment is hair bulb. The center of hair bulb is invigilated at the lower end to form the dermal papilla. These cells in contact with dermal papilla are called hair matrix. These are responsible for actual production of hair. It contains melanocytes responsible for color of hair. Fig. No. 1. Hair Shaft (

3 Journal of Biotechnology and Biosafety "#$%&'()*++%',)-./%.0123'40%.01(5,6)(,29, *778(9((2565: Journal of B iotechnology and B iosafety Hair shaft (Tortora G. J., Derrickson B. H, 2009, p-157) Figure no. 1 shows a longitudinal and transverse section through a hair shaft. The shaft and the root of the hair both consist of three concentric layers of cells; medulla, cortex and cuticle of the hair. Cuticle The cuticle is the outermost layer made of flattened cells arranged in an overlapping roof like pattern thickness of each cell is 0.5 µm. action. However, the treatments for dandruff offered at present do have an ability to reduce the vitality of the organism, but it is not clear whether this is their primary function. Types of Dandruff: (Virtue s Family Physician, 1981) Dandruff can be classified as disorders of the sebaceous gland or skin scaling disorders. Dandruff can be of two types, Oily dandruff Cuticle is also responsible for surface properties of hair, shine and smoothness. Cortex It forms the bulk of the hair and it consists of numerous layer of flattened elongated cells packed together. It gives strength elasticity to the hair. It also gives color to hair due to presence of pigment. Medulla Medulla is the innermost layer of hair. The presence or absence of medulla does not have too much to do with human hair behavior, when washed with water, on color or on curl it. Dandruff (Wilkinson J. B et al., 1990, p-367) Dandruff is a common condition among men and women of all ages, and often causes skin irritation, embarrassment and annoyance to sufferers. In consequence, it has been the subject of numerous investigations and the target of many forms of therapy. So far there has been little success in explaining or curing the disease. Definition: (Wilkinson J. B et al., 1990, p-17) The disorder of the skin characterized by massive flaking of the stratum corneum which continues to curiosity of cosmetic chemist, is dandruff. No clinical analogy exists for this condition and views as to its etiology come from many areas of interest. The disease is simply a microbial infection with the yeast Pityrosporum ovale. This lipophilic organism which resides on the stratum corneum of the human scalp has been studied extensively without conclusive evidence for its involvement in dandruff being forthcoming. The theory depends upon the secretion by the organism of a metabolite or waste products which enters the skin and invokes large scale flaking from the stratum corneum. Direct contact of the skin with the products of metabolism of the organism has failed to give conformation of this Dry dandruff Dry dandruff again can be mild or severe. Oily dandruff (Pityriasis Steatoides): On the scalp, waxy, greasy, yellowish, thick scales crusts are present. Beneath the crusts, the scalp is red or pale but dry. The hair may be dull and flat without shine. There may be slight itching. If irritated, eczematization complicates the condition to produce seborrhoeic dermatitis. Patients with pityriasis steatoides usually develop thing and later loss of hair. (Puri I, 1979). Dry dandruff (Pityriasis sicca): The scales are fine, thin, furfuraceous, white or grayish and dry or only slightly greasy. The hair is dry and lusterless. There is mild to moderate itching. The scales fall freely on the shoulders. This type of dandruff is more common in winter than in summer. It signifies exaggeration of normal exfoliation of the horny layer of the epidermis. It usually affects people with dry integument and scalp. In nutritional disorders, scaliness of the scalp is exaggerated. (Behl P. N, 1982). Etiology of dandruff: The causation of dandruff is still debatable and it seems undoubtedly that it is the sum total of many factors working simultaneously. The discussion about the cause of dandruff revolves around the relative roles of physiological traumatic and infective factors. (Wilkinson J. B et al., 1990, p-418 and Mitsui T, 1997) Some of the causes of dandruff are: Abnormal keratinization of the epidermal tissue Excessive lipid secretion Abnormal proliferation of scalp bacteria

4 Journal of Biotechnology and Biosafety "#$%&'()*++%',)-./%.0123'40%.01(5,6)(,29, *778(9((2565: Journal of B iotechnology and B iosafety The yeast like organism, Pityrosporum ovale and Pityrosporum orbicular are common members of the scalp flora. Pityrosporum ovale was first described in 1874 by Malassez, who believed it to be the role cause of dandruff. (Wilkinson J. B et al., 1990, p-158) Pityrosporum or Melassezia: (Woods G. L., Gutierrez Y, 1993) The genus Melassezia contains at least two species of globose to ellipsoid, unipolar, budding yeasts i.e. Pityrosporum ovale and Pityrosporum orbicular. The organisms bud repeatedly forms a broad base from some location, forming a collarette from which younger bud emerge. The pathogenesis of infections caused by Malassezia furfur is not completely understood. Other causes of dandruff: (Butler H, 2000) External factors: Biochemical changes of the cutaneous scalp. Increase in the number and activity of bacteria and fungi. Localized inflammatory reaction following the use of topical medications and cosmetics. Hormonal imbalance. Internal factors: Impaired metabolic nutrition. Dietary factors. Nervous tension. SHAMPOO Originally, shampoos were made up of soap or mixtures of soaps; today synthetic detergents are used in the majority of commercial products. (Balsam M. S., Sagarin E, 2008, p- 73). Definition Shampoo is defined as a preparation of a surfactant (i.e. surface-active material) in suitable form-liquid, solid, or powder- which when used under the condition specified will remove surface grease, dirt, and skin debris from the hair shaft and scalp without affecting adversely the hair, scalp, or health of user. (Balsam M. S., Sagarin E, 2008,p-74) In earlier, a shampoo was defined as a suitable detergent for the washing of hair, packaged in a form convenient for use. The Good housekeeping consumer panel found that women wanted a shampoo to clean and also to rinse out easily, impart gloss to the hair and leave it manageable and non-drying. (Wilkinson J. B., Moore R. J, 1990, p-398) Ideal properties of shampoo (Sharma P. P, 1994) It should remove soil, sebum and residues of hair setting lotions or dressing from hair and scalp. It should produce the foam of the degree that will satisfy the user. It should be easy to remove shampoo by rinsing leaving the hair soft, lustrous and manageable. It should impart a pleasant fragrance during use, masking the odour of wet hair. Ease of combing of the wet hair. This evaluates the roughness of the hair immediately after treatment with the detergent, and under conditions where this roughness is most apparent. The consumer associates this property with the cleansing action of the shampoo. It goes without saying that the shampoo detergent must be safe for use on the scalp, and no irritation reddening or other discomfort should be caused by its use. INTRODUCTION: Piroctone olamine (O Neil M. J et al., 2006) Piroctone olamine acts as anti-microbial agent. It is used as anti-microbial against various microorganisms including bacteria, fungi, mould, yeast etc. With reference to literature survey, in this present project work, an attempt has been made to evaluate the anti-fungal property of Piroctone olamine against Pityrosporum ovale, Candida albicans and Malassezia furfur. This is the main reason to take up this study INCI Name : Piroctone olamine Common Name: Piroctone olamine

5 Journal of Biotechnology and Biosafety "#$%&'()*++%',)-./%.0123'40%.01(5,6)(,29, *778(9((2565: Journal of B iotechnology and B iosafety Chemical Name: 1-Hydroxy-4-methyl-6-(2, 4, 4-trimethylpentyl) 2-(1H) pyridinone, 2-aminoethanol salt Empirical formula: Emp. Formula : C 14 H 23 NO 2 C 2 H 7 NO Mol. Weight : g/mol Structural Formula: Physical-chemical properties: Appearance : White or slightly yellow crystalline powder. Solubility : Freely soluble in 10% ethanol in water; soluble in solution containing Surfactant in water or in 1%- 10% ethanol; slightly soluble in water and in oil. PH : (1% suspension in water, 20 C) Melting range : C EXPERIMENTAL ANALYSIS OF PIROCTONE OLAMINE (ACTIVE): Physical And chemical analysis: The physical and chemical analysis of Piroctone olamine was carried out as directed in analytical report of Piroctone olamine. 1) ph (The Indian Pharmacopeia,2010, p-146): Apparatus: Beaker, ph meter, stirrer, wash bottle. Procedure: The ph meter was calibrated by using buffer solutions. 1% solution was prepared in distilled water and ph was determined by using ph meter. 2) Melting Point (The Indian Pharmacopeia,2010, p-140): Apparatus: Beaker, water bath, capillary tube, thermometer, theils tube Procedure: As per mentioned in Indian Pharmacopeia, procedure was followed and melting point was determined. 3) Solubility (The Indian Pharmacopeia,2010, p- 147): Apparatus: Test tube, beaker Procedure: As per mentioned in Indian Pharmacopeia, procedure was followed and solubility was determined. 4) Residue on Ignition (The Indian Pharmacopeia, 2010, p-87): Apparatus: Silica crucible, sulfuric acid, muffle furnace, weighing balance. Procedure: As per mentioned in Indian Pharmacopeia, procedure was followed and residue on ignition was determined.

6 Journal of Biotechnology and Biosafety "#$%&'()*++%',)-./%.0123'40%.01(5,6)(,29, J o u rna l o f B io techn olo gy an d B iosa fety *778(9((2565: Observation: Table no. 1- Observation for Residue on Ignition: Wt. of residue after each heating (in gm) Materials to be weighed Wt. (in gm) Sr. No. 1 Wt. of empty silica crucible Wt. of silica crucible + sample Wt. of sample taken Calculation: "#$%&"""#$%$&# "" """"# ""# "# "#$%&"""#$%$&# "# 5) % Purity: (as mentioned in standard testing procedure of Yasham Bio-science, Mumbai) Apparatus: Beaker, conical flask, volumetric flask, burette, stirrer. Procedure: 0.3 g of Piroctone olamine was weighed and dissolved in 50 ml of ethanol and 20 ml water then titrated with 0.1 M sodium hydroxide, by using phenol red solution as indicator, until a reddish violet color was obtained. Then by using 1 ml of 0.1 M NaOH is equivalent to g of Piroctone olamine this factor, the percentage purity was determined. Observation: Table no. 2- Observation for Percentage Purity: Sr. No. Materials to be weighed Wt. (in gm) 1 Wt. of butter paper Wt. of butter paper + sample Wt. of sample taken Burette reading (B.R.) = 23.1 ml Calculation: "#$%& "#$""" Where, B.R. Burette reading N Normality of NaOH W Weight sample taken ""#"#$""" "" "## "## "#$%& "

7 Journal of Biotechnology and Biosafety "#$%&'()*++%',)-./%.0123'40%.01(5,6)(,29, *778(9((2565: Journal of B iotechnology and B iosafety ANTIMICROBIAL ANALYSIS OF PIROCTONE OLAMINE: (Dubey R. C., Maheshwari D. K.; Practical Microbiology, 2005, p-154, 155) The active ingredient Piroctone olamine was subjected to invitro testing by using agar-diffusion method to evaluate its anti-fungal activity against Candida albicans, Malassezia furfur and Pityrosporum ovale. The cultures of above organisms were procured from Rajiv Gandhi Institute of Bio-Technology, Nagpur. The cultures were sub-cultured and grown in suitable growth media. Preparation of media: For Candida albicans and Pityrosporum ovale: The culture of Candida albicans and Pityrosporum ovale were cultivated and maintained on a growth medium described below. The incubation was done at room temperature for 48 hours. Potato dextrose agar (PDA): 39 gm of PDA and 5 gm agar-agar were weighed and transferred to 1000 ml borosil beaker to this 1000 ml of distilled water was added and was stirred on water bath for 20 minutes until frothing appears during heating. Then sterilization was done in autoclave at 121 C and 1.05 kgf/cm 2 pressure for 20 minutes. After sterilization it was kept in refrigerator. For Malassezia furfur: The culture of Malassezia furfur was cultivated and maintained on a growth medium described below. The incubation was done at room temperature for 7 days. Sabouraud dextrose agar (SDA) with corn oil: 30 gm of SDA and 5 gm agar-agar were weighed and transferred to 1000 ml borosil beaker to this 1000 ml of distilled water was added and was stirred on water bath for 20 minutes until frothing appears during heating. Preparation of solution of Active ingredient: 1 gm of sample which is to be analyzed was weighed to make 1% solution in sterile distilled water. Media used for screening: For Candida albicans and Pityrospum ovale, Potato Dextrose Agar was used. For Malassezia furfur Sabouraud Dextrose Agar with corn oil was used Procedure: 1. All the apparatus and media were sterilized in an autoclave at 121 C and 1.05 kg/cm 2 pressure for 20 minutes. 2. The sterile and melted media were cooled at 45 C, then poured in labeled Petri plates and allowed to solidify. 3. After solidification of media, the pure culture of Candida albicans, Malassezia furfur and Pityrosporum ovale were spread on the solidified media for respective species. 4. The wells were made in each labeled plate with the help of sterile cork borer. 5. Previously prepared 1% sample solution of active was poured in different labeled Petri plates. For each species one plate was kept as positive control, in which only distilled water was poured. 6. Then all plates were incubated at 37 C for 48 hours 7. After incubation, the zones of inhibition were measured in mm of each species. RESULT AND DISCUSSION: Common results of analysis Piroctone olamine: Table no. 3- Analytical test observation for Piroctone olamine: Sr. Test Specification Results Inference No. 1 Colour White to Creamish Complies Passes test 2 Odour To match Std. Complies Passes test 3 ph (1% Solution) Passes test 4 Melting Point C C Passes test 5 Solubility 6 Residue on Ignition (%) Freely soluble in 10% ethanol, Slightly soluble in water, completely soluble in surfactant solution Clear Solution Passes test Max % Passes test 7 Purity (%) % 99.49% Passes test

8 Journal of Biotechnology and Biosafety "#$%&'()*++%',)-./%.0123'40%.01(5,6)(,29, *778(9((2565: Journal of B iotechnology and B iosafety Anti microbial activity observation: The zone of inhibition of Piroctone olamine against Candida albicans, Malassezia furfur and Pityrosporum ovale were measured and are given in table no.4 Table no. 4- Antimicrobial test observation for zone of inhibition (in mm): Sr. Microorganism Concentration of Active No. 0% (Blank) 1% 1 Candida albicans (Fig 1,2,3,4) 00 mm 27 mm 2 Malassezia furfur 00 mm 20 mm 3 Pityrosporum ovale 00 mm 24 mm Zones of Inhibition of Active against Candida albicans: Before Incubation After Incubation Fig 1: Distilled Water (Blank) Fig 2: Distilled Water (Blank) Fig 3: 1% Active Solution Fig 4: 1% Active Solution Zones of Inhibition of Active against Malassezia furfur: Before Incubation After Incubation Fig 5: Distilled Water (Blank) Fig 6: Distilled Water (Blank) Fig 7: 1% Active Solution Fig 8: 1% Active Solution

9 Journal of Biotechnology and Biosafety "#$%&'()*++%',)-./%.0123'40%.01(5,6)(,29, *778(9((2565: Journal of B iotechnology and B iosafety Zones of Inhibition of Active against Pityrosporum ovale: Before Incubation After Incubation Fig 9: Distilled Water (Blank) Fig 10: Distilled Water (Blank) Fig 11: 1% Active Solution Fig 12: 1% Active Solution CONCLUSION: Every individual wishes healthy hair on the scalp which plays a very important role in the social and personal life. From this study we could understand the structure of hair, the types of dandruff and the types of shampoo. It is very important to understand the type of hair one possess and respectively the choosing of the shampoo. Here is a small attempt to understand the same which is helpful for everyone to maintain the hairs on the scalp in a better way. From above observation it was concluded that, Piroctone olamine has excellent anti-dandruff activity against above dandruff causing species which can be used in the anti dandruff shampoo formulation. REFERENCES: Balsam M. S., Sagarin E.(2008). Cosmetic Science and Technology. 2 nd Edition. 2:73 Balsam M. S., Sagarin E.(2008). Cosmetic Science and Technology. 2 nd Edition. 2:74 Behl P. N. (1982). Practice of Dermatology. 5 th Edition. Pg. No. 419 Butler H., Poucher s (2000). Perfume, Cosmetics & Soap; 10 th Edition. Kluwer Academic Publishers, London; Pg. No.580 Dubey R. C., Maheshwari D. K. (2005) Practical Microbiology. 1 st edition. S. Chand and Company Ltd.; Pg. No. 154,155 Government of India, Ministry of Health and Family Welfare. Indian Pharmacopeia, 2010; Vol. 1. The Indian Pharmacopeia Commission, Ghaziabad; Pg. No. 82

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