Characterization of Normal and Dandruff Causing Micro-Flora from Human Scalp of Various Age Groups

Ms. Anuya R. Belekar, Prof. Savanta V. Raut

Department of Microbiology, Bhavan’s College, Munshi Nagar, Andheri (W), Mumbai- 400058.

*Corresponding Author E-mail: svrmicro@yahoo.co.in

ABSTRACT:

Dandruff is the most common problem that affects many people leading to an embarrassing condition. It results from three main factors namely: Malassezia fungi, sebaceous secretion and individual sensitivity. To study the characterization of dandruff causing micro-flora from healthy and affected scalp sample according various age groups (1-10, 10-30, 30-60 and above 60) scalp sample of healthy and affected scalp sample were cultured in appropriate media like Nutrient agar, Sabouraud’s dextrose and Dixon’s agar. The normal micro-flora (including Yeast and Bacteria) from the scalp with dandruff was compared to the flora from people without dandruff. The bacterial and fungal communities associated with dandruff were investigated using culture-dependent Method. Normal Bacterial count of healthy and dandruff-affected scalp sample of various age group (1-10, 10-30, 30-60 and above 60) were 0.003, 0.2549, 2.0082, 1.1800 and 1.17, 1.0909, 1.2554, 0.7729 respectively. Normal Yeast count of healthy and dandruff-affected scalp sample of various age group (1-10, 10-30, 30-60 and above 60) were 0.03, 0.0014, 1.3202, 0.8987 and 0.016, 0.0034, 0.6692, 0.9117 respectively. Malassezia count of healthy and dandruff-affected scalp sample of age group (1-10, 10-30, 30-60 and above 60) were 6.6, 100.2, 62.6 and 61.4,300, 63.2 respectively. Malassezia count was absent in age group (1-10) of both scalp sample. Bacterial and Yeast count of dandruff affected scalp were increases in age group (1-10, 10-30) as compare to healthy scalp sample. But in age group (30-60 and above 60) normal Bacterial and Yeast count was decrease in dandruff-affected scalp sample. Malassezia furfur and Malassezia dermatis were identified by Biochemical test method. A comparative study on the efficacy of chemical and herbal anti-dandruff ingredients on basis and their performance in market shampoos was done in vitro against normal micro-flora including (Bacteria, Fungi and Malassezia Species.) which found on healthy and dandruff-affected scalp sample. Banana extract (Musa Acuminata) were also tested against normal micro-flora and Malasssezia Species. Banana extract showed excellent anti-malassezia activity against Malassezia Species. But failed to show antibacterial and anti-fungal activities. Zone of inhibition of herbal and banana extract showed lower value than synthetic (Chemical shampoo) ingredients containing ZnPTC and Ketoconozole recorded higher in vitro activities than shampoos containing herbal AD ingredients and Banana extract.

KEYWORDS: Dandruff, Age groups, Chemical and Herbal shampoo, Banana extract.

1. INTRODUCTION:

Dandruff is a common chronic scalp condition marked by flaking of the skin on scalp. Dandruff (dandruff, dandriffe) is the popular word of Anglo-Saxon origin, a combination of ‘tan’ meaning ‘tatter’ and ‘drof ’ meaning ‘dirty. Dandruff is a common scalp disorder affecting almost half of the population at the pre-pubertal age and of any gender and ethnicity. In Dandruff, the flakes are loosely adherent, oily generally not associated with overt inflammation, and restricted to the scalp. Dandruff particles are visible flakes of skin that have been continuously shed from the scalp. It is normal to shed some dead skin flakes as the skin is constantly renewing itself. The characteristic flaking and sealing of the scalp suggest impairment in the desquamation process. Dandruff has been shown to possibly be the result of three factors:

* Sebum production

* Microbial metabolism

* Susceptibility of individual.

Sebum keeps the hair healthy and smooth and it keeps the hair from excessive drying and from breaking. Sebaceous gland activity shows strong association with most disorders that cause scalp flaking. Early sebum production in the neonate may manifest as cradle cap. Once maternal hormone control subsides, sebum production does not begin again until puberty, at which time sex hormone control affects sebum production. As sebum production increases, Malassezia proliferates in response to new food sources. Proliferation of Malassezia increases lipid metabolism, which causes scalp itching and flaking. Seborrheic dermatitis, a condition in which an excessive amount of sebum is produced and builds up on the scalp (looking like an adult cradle cap) is also a symptom of hormonal imbalances, as is an excessively oily or dry scalp. Both can cause hair thinning.

This correlation includes increased incidence during infancy (cradle cap), low incidence from infancy to puberty, an increase in adolescence and young adulthood, and a decrease later in life. In addition, dandruff occurs exclusively on skin in areas with high levels of sebum. Malassezia (formerly known as Pityriasis) is a genus of related fungi. These yeasts are naturally found on skin surfaces of many animals including man. Malassezia consists of at least 11 species that is M. furfur, M. globosa, M. restrica, M. pachydermatis, M. obtusa, M. slooffiae, M. sympodialis, M. japonica, M.nana, M. dermatis, and M. yamatoensis) out of this M .pachydermatis are non lipid-dependent species. During dandruff, the levels of Malassezia increase by 1.5 to 2 times its normal level. The normal microflora which is present on healthy scalp is Micrococcus luteus, P.acnes, staphylococcus aureus, streptococcus pneumonia,Brevibacterium species, Trichophyton tansurans, Microsporum audouinii ,Microsporum canis, Trichophyton violdeeum, Trichophyton schoenlenii, Filobasidum spps, Acremonium spps,cornebacterium acnes, Lice ,ticks (Park S-G, Kim MN, Kim BJ, et al. 2012).

Genetic, physiological factors, environmental, hormonal and immune system factors have been shown to be involved in the manifestation of Seborrhoeic dermatitis. Seborrhoeic dermatitis may be aggravated by illness, physiological stress, fatigue or sleep deprivation.Diet is not the direct cause of this problems, poor diet, especially deficient in vitamin B can make it worse. The use of inadequate shampoos, fall of detergents or use of hair laequeres or hair dyes increase dandruff. Similarly, washing or brushing your hair too hard can encourage peelings. Environmental factors are living in low humidity places promotes the growth of dandruff. Common symptoms of dandruff in adults are itching, oily flakes in scalp, tingly feeling scalp, red flaky dandruff and, in rare cases, crusting and scaling rash. A severe case of dandruff leads to facial dry skin, ear eczema that keeps reoccurring, chest rash with red spots and rash in eyebrows, nose, and ears.

Many synthetic antifungal preparations (miconazole, ketoconazole, selenium sulfide, salicylic acid, coal tar, zinc pyrithione) have been tested against Malassezia species, but the frequent infections and the recurrences have made the therapy costlier. Patients uncomfortable or unsatisfied with synthetic pills and prescriptions may consider turning to herbal remedies extracted from plants, roots, seeds and fruits. Banana is the common name for herbaceous plant of genus Musa acuminate. Some fruits like bananas offer great medical benefits. Banana are great for hair and scalp. They improve manageability and shine while moisturizing skin and helping to prevent and control dandruff. Banana is rich in potassium, natural oils, carbohydrates and vitamins. These help in softening the hair and protects the hair’s natural elasticity preventing split ends and breakage. Banana when used for hair has a number of benefits such as creates manageability, shine, growth and controls dandruff. These are simply the best for hair and scalp.

2. MATERIALS AND METHODS:

This study was conducted in the departments of Microbiology, Bhavan’s College, Mumbai, India. 20 dandruff-volunteers (5 each from various age groups 1-10, 10-30, and 30-60, above 60) and 20 healthy individuals (5 each from various age groups 1-10, 10-30, 30-60, above 60) without features of dandruff (controls) were enrolled in this study. The study was conducted during August 2014 to March 2015.

2.1) Sample collection:

(Roia. C. Frank, Vanderwyk. W. Raymond, 1969)

Based on the presence or absence of visible flakes over the scalp, the study population was categorized as dandruff-affected patients or healthy individuals, respectively. The dandruff volunteers were defined as individuals having inflammation of the scalp in the form of red, scaly, itchy and flaking rash. The samples were collected after obtaining written informed consent of the volunteers. Sampling was carried out at convenience by random screening for the dandruff cases in the selected age groups. 5 scalp sample were collected from infected person and healthy person of various age group (0-10, 10-30, 30-45 and 45 onwards) by using sterile swab and sterile scalpel were used for scratching the scalp. Samples were collected from relatively severely affected areas of the patients. Flakes or scales were collected by scrapping approximately one inch area using blunt scalpel. A uniform sample of scalp were collected by making several passes over the scalp in a left to right manner from the temple and forehead to the nape of the neck. Sterilize swab by autoclaving at 15 lbs. pressure (121⁰c) for 15 minutes. A mixture of ethylene oxide (10%) and carbon dioxide (90%) were used for sterilization for scalpel. Then swab sample were transfer in 2ml of sterile saline. In healthy individuals, the scrapings were collected from vertex and temporal regions. Each sample was labeled with the volunteer’s name. Both children and adults were included in the study. On the day of sampling, the participants were advised not to wash their scalp.

2.2) Observation on scalp samples:

(Roia .C.Frank,Vanderwyk.W.Raymond,1969)

Collected scalp sample was spread on clean, grease free slide and Gram stain it.

2.3) Micro-flora analysis:

(Shu’aibu. Issa et al, 2004)

Sample which collected from healthy and infected person will be plated on basal synthetic media.

2.3.1) Quantitative analysis:

(Shu’aibu. Issa et al, 2004)

Make dilution of scalp sample up to 10^-1,10^-2,10^-3 and take 0.1ml of 10^-2,10^-3 and plated on Nutrient agar (For Bacteria), Sabouraud’s dextrose agar (For Mold and Yeast).Poured 20ml of molten Nutrient agar onto empty plate. Allow to solidified and then plated onto Nutrient agar plate and Sabouraud’s dextrose agar.

2.3.2) Detection and Isolation of Malassezia Species: (Shu’aibu Isa et al, 2004)

Swab the undiluted scalp sample on selective media (Dixon’s media containing chloramphenicol and cycloheximide). Poured 20ml of molten Dixon’s agar onto empty plate. Allow to solidified and then plated or swab onto Dixon’s agar plate.Chloramphenicol is Broad spectrum antibiotic widely used against Gram positive and Gram negative bacteria. Cycloheximide is an antibiotic which is very active against many molds, yeasts, and phytopathogenic fungi. Malassezia species are resistance to the both of this antibiotics.

2.3.3) Identification of Malassezia Species:

2.3.3.1) Bio-chemicals:

The organism was biochemically analysed by using catalase test, splitting of esculin, Urease test.

2.3.3.2) Catalase reaction: (Ahmad. W, Karhoot. J, Noaimi.A, 2012)

Presence of catalase is determined by using a drop of hydrogen peroxide (3% solution) and production of gas bubbles is consider as a positive reaction.

2.3.3.3) Splitting of Esculin: (Ahmad. W, Karhoot. J, Noaimi.A, 2012)

An inoculum from a pure culture was transferred aseptically to a sterile tube of bile esculin agar and streaked along the slant. There is no need to stab the medium. The inoculated tube was incubated at 32⁰ C for 5 days.. The splitting of esculin is revealed by darkening of the medium. This test was use to distinguish M. furfur, M. slooffiae and M. sympodialis from other Malassezia species.

2.3.3.4) Urease test: (Ahmad. W, Karhoot. J, Noaimi.A, 2012)

An inoculum from pure culture was transferred aseptically to a sterile tube of urease broth. The inoculate tube was inoculated at 32⁰ for 24 hrs. The presence of urease test indicated by pink color of media.

2.3.3.5) Gram’s Staining: (Sujogya K.P., 2012)

A smear of the pure culture obtained after two weeks of incubation at 32 ⁰C and gram stained it.

2.3.3.6) Statistical Analysis: (Clavaud C, Jourdain R, Bar-Hen A, Tichit M, Bouchier C, et al. ,2013)

Z-test and Paired t-test was used for statistical analysis in this study for distribution of Micro-flora in relation to different age groups and Normal and Dandruff -affected human scalp.

2.4) To check antifungal activity of Alcoholic extract of banana leaves against Malassezia Species:

2.4.1) Collection of plant material:

(B. Meenashree, V.J.Vasanthi, 2014)

The Banana leaves were collected from the campus of Bhavan’s College of botanical garden. The collected leaves were washed thoroughly under running water and air dry for few minutes. The fresh leaves were immediately extracted with the solvents.

2.4.2) Preparation of leaf extracts:

(B.Meenashree, V.J.Vasanthi, 2014)

Take a 10 grams of fresh finely cut banana leaf and add 50ml of acetone (96%) separately. It was incubated for 3 days on shaker and then filtered it. After filtration, the supernatants were evaporated in evaporating dish to obtain the crude extract. The extracts are suspend in 10ml of acetone and then syringe filter it. This was used as the stock solution.

2.4.3) Disc Diffusion Method:

(Arumugam.P, Nirmala Nithya, 2013)

Diffusion dependent activities of the acetone extract of banana leaves was studied by zone of inhibition. The 48 hours broth culture is uniformly swab onto the surface of the Sabouraud’s dextrose medium (yeast), Dixon’s agar medium (Malassezia). All chemical and herbal shampoos (CS-1, CS-2, CS-3, HS-1) were dissolved in sabouraud’s broth at 1:5, 1:10 concentration and the undiluted acetone extract of banana leaves were used. The disc dispensed in different concentration of chemical and herbal shampoos and undiluted acetone extract of banana leaves and placed on the Petri plate seeded with organisms. The plates were incubate at 37°C for 24 hours and the zone of inhibition was measured. The antifungal activity of acetone extract of banana leaves were compare with Nystatin disc (for fungal) and 3% Sulfur powder (for Malassezia), because of their well established activity against this species. (Ranganathan S et al., 2010)

2.4.4) Agar cup method:

(Arumugam.P, Nirmala Nithya, 2013)

A standardize inoculum culture was added into 20 ml of molten Muller and Hintons agar and then poured onto empty sterile petri plate then spread evenly and allowed it for solidified. Made Wells of 8 mm were aseptically punch on the agar using a sterile cork borer allowing at least 30 mm between adjacent wells and the Petri dish. Various concentration of dilution were prepared (1:5, 1:10). 50 ul of acetone extract of banana and chemical and herbal shampoos of various dilution were then introduced into the wells (plate-2). The plates were then incubated at 32⁰C at 24hrs and the zone of inhibition was measured.

3. RESULTS AND DISCUSSION

3.1) Bacterial, yeast and mold, Malassezia count of (1-10, 10-30, 30-60, above 60) age groups:

5 scalp sample was collected from infected person and healthy person of various age group (0-10, 10-30, 30-60,60onwards) by using sterile swab. Culture were spread on Nutrient agar (for bacteria), Sabourauad’s agar (for fungi and yeast) and Dixon’s agar media (for Malassezia spps.). Bacteria, fungal and Malssezia count of scalp sample of various age groups are as Table No.3.1 to 5.8 and Fig.No.3.1, to 3.25

Table No 3.1: Age Group (1-10) year old (Healthy)

Sample No.	Types Of Sample(Healthy)
	Bacterial Count (ml)		Yeast And Mold (ml)		Malassezia Species.
	Average and Standard Deviation	Z-Test	Average and Standard Deviation	Z-Test
1(1years/F)	3.5×10^3± 0.310	0.645	3.5×10^4± 0.208	0.961	-
2(2years/M)	2.8×10^3± 0.583	1.200	3.5×10^4± 0.244	1.598	-
3(5years/F)	3.1×10^3± 0.16	1.25	3.4×10^4± 0.16	1.25	-
4(7years/F)	3×10^3± 0.41	1.219	3.5×10^4± 0.208	1.442	-
5(10years/F)	2.7×10^3± 0.141	2.127	3.7×10^4± 0.25	1.200	-

Table No 3.2: Age group (1-10) year old (Affected)

Sample No.	Types Of Sample(Affected)
	Bacterial Count (ml)		Yeast And Mold (ml)		Malassezia Species.
	Average and Standard Deviation	Z-Test	Average and Standard Deviation	Z-Test
6(3years/M)	2.408×10^6± 3.282	1.642	0.602×10^3± 0.7561	1.848	-
7(5years/F)	1.107×10^5± 1.583	1.827	0.25×10 ^3± 0.375	2	-
8(5years/F)	1.679×10^6± 2.6716	1.729	-	-	-
9(8years/F)	0.83×10^6± 1.1145	1.947	0.825×10^6± 1.1187	1.944	-
10(9years/F)	0.847×10^6± 1.1000	1.957	0.825×10^4± 1.1187	1.944	-

Table No 3.3: Age group (10-30) year old (Healthy)

Sample No.	Type of sample (Healthy)
	Bacterial Count (ml)		Yeast And Mold Count (ml)		Malassezia Species
	Average And Standard Deviation	Z-Test	Average And Standard Deviation	Z-Test
1(22 Years/F)	4.295×10^5± 4.205	1.000	0.5×10^4± 0.35	1.428	8
2(22 Years/F)	3.447×10^5± 3.253	0.3372	1.8×10^4± 1.2	1	1
3(23Years/F)	3.37×10^5± 2.73	1.000	0.5×10^3± 0.35	1.428	-
4(24Years/F)	4.3×10^4± 1.7	1.000	0.5×10^3± 0.35	1.428	1
5(25 Years/F)	1.203×10^5± 1.097	1.000	4×10^3± 2.828	1.414	23

Table No 5.4: Age group (10-30) year old (Affected)

Sample no.	Type of sample (Affected)
	Bacterial count(ml)		Yeast and mold count (ml)		Malassezia species
	Average and standard deviation	z-test	Average and standard deviation	z-test
6(11Years/M)	1.65×10^6± 1.35	1	0.5×10^4± 0.7071	1.131	-
7(12Years/F)	0.725×10^6± 0.425	1	1×10^3± 0.707	1.414	3
8(16Years/F)	1.02×10^6±0.72	1.791	1.5×10^3± 1.5	1	3
9(20Years/M)	1.65×10^6±1.35	1	1.65×10^6±1.35	100	1
10(22Years/F)	4.095×10^5± 3.905	1	0.5×10^3± 0.35	1.428	300

Table No 5.5: Age group (30-60) year old (Healthy)

Sample No.	Type Of Sample (Healthy)
	Bacterial Count (ml)		Yeast And Mold Count (ml)		Malassezia Species
	Average and Standard Deviation	Z-Test	Average and Standard Deviation	Z-Test
1(35years/F)	2.35×10^6± 2.05	1	3.525×10^5±2.475	1	14
2(39years/F)	0.8×10^6± 0.5	1	2.66×10^6±2.36	1	23
3(40years/F)	3.73×10^6±0.77	1	2.615×10^5±1.785	1	300
4(44years/F)	2.23×10^6±1.93	-1	2.524×10^6±1.956	1	136
5(44years/F)	0.931×10^6±0.519	1	0.803×10^6±0.347	1	28

Table No5.6: Age group (30-60) year old (Affected)

Sample No.	Type Of Sample (Affected)
	Bacterial Count (ml)		Yeast And Mold Count (ml)		Malassezia Species
	Average and Standard Deviation	Z-Test	Average and Standard Deviation	Z-Test
6(35years/F)	2.48×10^6± 2.13	1.258	1.69×10^6±1.39	1	-
7(37years/F)	5.02×10^5± 2.98	1	0.5×10^4±0.35	1.428	300
8(38years/M)	1.65×10^6±1.35	1	1.65×10^6±1.35	1	300
9(45years/F)	0.755×10^6±0.455	1	0.5×10^3±0.35	1.428	-
10(48years/F)	0.89×10^6± 0.59	1	0.5×10^3±0.35	1.428	300

Table No 5.7: Age group (above 60) year old (Healthy)

Sample No.	Type Of Sample (Healthy)
	Bacterial Count (ml)		Yeast And Mold Count (ml)		Malassezia Species
	Average and Standard Deviation	Z-Test	Average and Standard Deviation	Z-Test
1(62Years/M)	0.96×10^6± 0.66	1	1.65×10^6± 1.35	1	4
2(63Years/M)	3.25×10^6± 2.95	1	1.13×10^6± 0.83	1	300
3(64Years/F)	1.581×10^4± 1.419	1	2.935×10^4± 2.165	1	1
4(65Years/F)	2.425×10^4± 1.675	1	3.43×10^4± 2.37	1	1
5(75Years/F)	1.65×10^6± 1.35	1	1.65×10^6± 1.35	1	7

Table No 5.8: Age group (above 60) year old (Affected)

Sample No.	Type Of Sample (Affected)
	Bacterial Count (ml)		Yeast And Mold Count (ml)		Malassezia Species
	Average and Standard Deviation	Z-Test	Average and Standard Deviation	Z-Test
6(60 Years/F)	0.87×10^6± 0.57	1	0.598×10^6± 0.422	1	4
7(62 Years/F)	4.95×10^5± 1.95	1	1.65×10^5± 1.35	1	7
8(63Years/F)	4.7×10^5± 2.8	1	3.08×10^5± 2.02	1	3
9(64Years/F)	1.65×10^6± 1.35	1	1.65×10^6± 1.35	1	300
10(65Years/F)	3.795×10^5± 1.305	1	3.525×10^5± 2.275	1	2

TABLE NO 3.9: BIOCHEMICAL RESULT OF ISOLATED ORGANISM ON DIXON’S MEDIA

Biochemical tests
Organisms	Urease activity	Bile esculin	Catalase activity
IS-1	-	-	+
IS-2	+	+	+
IS-3	-	-	+
IS-4	+	+	+
IS-5	-	-	+
IS-6	-	-	+
IS-7	+	+	+
IS-8	+	+	+
IS-9	+	+	+
IS-10	-	-	+
IS-11	-	-	+
IS-12	-	-	+
IS-13	-	-	+
IS-14	-	-	+
IS-15	+	+	+
IS-16	-	-	+
IS-17	+	-	+
IS-18	+	+	+

Keys: ‘+’= Positive test, ‘-’= Negative test, ‘V’= Variable, ‘+’= positive or negativeFor Bile Esculin: ‘+’ = Weakly Positive test, ‘++’= Medium positive test, ‘+++’=Strongly Positive test

TABLE NO.3.10: Antimicrobial Susceptibility Testing of Banana Leaf Extract and Chemical Shampoos against Bacterial Counts

Antidandruff agent	Mueller and Hinton agar FOR BACTERIA				Mueller and Hinton agar FOR YEAST AND MOLD				Mueller and Hinton agar FOR MALASSEZIA ISOLATE
	BI-1	BI-2	BI-3	BI-4	YI-1	YI-2	YI-3	YI-4	MI-1	MI-2	MI-3	MI-4	MI-5
Banana extract	8	14.5	12	13	-	-	-	-	-	30	-	34	25
CS1
1:5	22	14.5	28.5	12	-	18.5	17	-	33	33.5	13.5	35.5	17.5
1:10	17.5	13.5	26	11	-	14	18	-	25	33	8	33	15
CS2
1:5	21.5	12.5	25	17	-	18.5	26.5	-	34	22	16.5	35.5	30
1:10	19.5	11.5	26	15	-	18	19.5	-	30	21	9.5	33	29
CS3
1:5	23.5	22	22.5	14.5	-	17.5	35	-	33	33	-	33.5	30.5
1:10	21	18.5	24	12	-	14.5	29	-	30	30	-	30.5	24.5
HS1
1:5	9	18.5	15.5	-	-	12.5	25	-	19	27	-	20	17
1:10	7	15	14	-	-	11.5	22	-	15	24	-	15	18
Control	19	-	18	-	-	-	22	-	12	14	-	20	-

Result shows that normal bacterial and yeast counts of age group (1-10 and 10-30) were increased in dandruff-affected scalp sample. During dandruff, loosely scaling produce large physical area so that large quantity of microbial flora can grow. Another contributing factor is by product of keratine product can influence microbial flora. Although this can be just a consequence of higher bacterial and fungal counts in the dandruff scalps, it can also indicate a higher propensity for the scalp of dandruff subjects to be colonized by a wide variety of scalp microbial contaminants. (Ce´Cile Clavaud, 2012). In age group (30-60 and above 60), Normal micro-flora of bacteria and yeast are decreased in dandruff-affected scalp sample. Because nutritional environment on some scalps is favorable for the growth of certain microorganisms. These suggested metabolic factors are: hormonal in-balance, impaired metabolism and nutrition, dietary indiscretions, as excessive carbohydrate and lipid intake, increased nervous tension; and biochemical changes of the cutaneous scalp (Frank c. Roia, 1969). Other metabolic factors reported by Spoor are the condition of the epidermis which may be a function of the individual's genetic makeup, his sex, age, and race and the amount and quantity of the sebum production (Frank c. Roia, 1969). Dandruff is due to an increase in the entire resident microbial flora, with each member having similar nutritional requirements (Frank c. Roia, 1969). In age group (1-10) Malassezia spps was absent in both scalp sample. But in age group (10-30, 30-60 and above 60) Malassezia count of dandruff-affected scalp sample was drastically increase than Malassezia count of healthy scalp sample. On dandruff-affected scalp, the level of Malassezia increases by 1.5-2 times their normal level. (Hee kuk park, 2012). Malassezia count was showed highest in age group of 30-60 followed by above 60 and 10-30 age group. In this study Malassezia count was found highest in age group (30-60 and above 60) because of androgen level. The incidence of the disease has two peaks: one in newborn infants up to three months of age, and the other in adults of around 30-60 years of age. The bimodal presentation of the disease (at birth and postpuberty) suggests that it may be associated with the sex hormones. Men are affected more often than women in all age groups and there is no preference for any specific ethnic group. (Sampaio ALB, Mameri A, 2011)

Malassezia count also observed 61.4 in age group (10-30). This because of Human scalp is very androgen sensitive and sebum rich. The sebum forms an ideal nutrient in the biocenose and sebum formation starts with the onset of puberty. However, sebum excretion rate in dandruff infected and non-infected subjects was found to be same. Many subjects with oily scalp did not show dandruff as well. This clearly shows that lipids may facilitate to some extent but not be the primary cause. It was noted that detection of different ratio of Malassezia species in patient with dandruff duto to the characteristics of patients. (Hee kuk park, 2012). Both in dandruff and seborrhoeic dermatitis, the population of Malassezia is not uniform throughout the skin surface and inside the stratum corneum.

In this study, Malssezia count was highest in healthy and dandruff-affected scalp than yeast and bacteria count. Increase in the total microbial flora was a factor in the increase of dandruff production since almost twice as many species of organisms were found in the scalps of those subjects with dandruff than on the scalps of those subjects without dandruff. (Roia, 1969) The average number of microbial species per individual is higher and the prevalence of specific groups of organisms is uniformly greater on the scalps of persons with dandruff than on those persons without dandruff. It is also believed that once dandruff is established an increase in the microbial flora will follow. This increase in microbial flora may contribute further to the dandruff condition as an additional factor regardless of original etiology.

Bacterial count of each age group (1-10, 10-30, 30-60, above 60) was higher than the normal yeast and mold count of healthy scalp sample, but this normal bacterial count of each age group (1-10, 10-30, 30-60, above 60) was lower than normal Malassezia count of healthy scalp sample. Malassezia count of healthy scalp sample of each age group was higher than normal yeast count of healthy scalp sample. In age group (1-10) Malassezia count was absent. Yeast count of dandruff-affected scalp sample of each group was higher than bacterial count of affected scalp sample, but this bacterial count of dandruff-affected scalp sample of each group was lower than Malassezia count. Malassezia count was absent in age group (1-10). Malassezia count of each age group was higher than yeast count present in dandruff-affected scalp sample.

The highest percentage 29% of bacterial count was found in age group 30-60 followed by 27% in 1-10 age group, 26% in 10-30 age group, 18% in above 60. Similar result were shown in Roia (1969) study, Resident bacteria were found in 57.1% of subjects with dandruff and in 25.0% of those without dandruff.

Percentage of yeast count was 1% in 1-10 age group, 0% in 10-30 age groups, 50% in 30-60 age groups, and 40 % in above 60. Percentages of yeast and mold count of dandruff-affected scalp sample of age group (1-10) were 9%, 0% were in age group (10-30), 39% were in age group( 30-60) ,52% were in above 60 age group. Roia study showed similar result, Yeasts were found in 56.4% of the subjects with dandruff and in 29.2% of those without dandruff. The average number of species of molds isolated from subjects with dandruff was 3.1, compared to 2.3 from subjects without dandruff. (Roia, 1969).Normal Malassezia count in age group 10-30 was 4% in healthy scalp while 14% in dandruff-affected scalp. In (30-60) age group, Malassezia count of healthy scalp sample was 59% and 71% in dandruff-affected scalp sample. In above 60, Malassezia count of healthy and dandruff-affected scalp sample was 37% and 15% respectively. Malassezia furfur was found in 96.4% of the subjects with dandruff and in 75.0% of the subjects without dandruff No specific organism, with the possible exception of P. ovale, showed any significant relationship to dandruff. (Roia, 1969).The present data show that in contrast to other studies dandruff is not only associated to the higher incidence of one particular Malassezia species but also to differences in the balance between the fungal and bacterial populations on the scalp.

3.2) Identification of isolate selected from Dixon’s media by Biochemical method.

18 organisms showing different characteristics was selected from Dixon’s agar plate. Identified Malassezia species by performing Bile esculin, Urease and Catalase test. [Table No.3.9 and Fig.No.3.26 and 3.27]

Out of 18 colony 7 organism showed same characteristics as Malassezia spps. Sample 2, 4, 7, 8, 9, 15 showed urease, bile esculin and catalase test positive. This sample showed similar colony characteristic (Mat, dull, smooth, convex elevation and oval, cylindrical shape) and biochemical test as M.furfur (sample 18 kept as control), while sample 17 were showed only urease and catalase test positive. This sample didn’t showed positive bile esculin test. Therefore this sample (sample 17) have similar colony characteristics (Convex, Butyrous, entire or lobed Margin and spherical, cylindrical shape) and biochemical test as M.dermatis. Sample 18 were kept as control.

M. restricta and M. globosa were the two most prevalent species isolated from dandruff patients,India. In Japan and USA, M. globosa is most frequently isolated from dandruff cases followed by M. restricta. Whereas M. furfur and M. slooffiae are isolated from scalp of healthy individuals and are considered as commensal species. (Rudramurthy et al, 2012)

Nakabayashi et al. reported having found 35% of M. furfur and 22% of M. globosa in individuals with SD. Rendic et al. found M. globosa in 67%, followed by M. furfur and sympodialis. Gupta and Gaitanis reported a greater amount of M. globosa. Tajima was the only author to report M. restricta as being the most common. (Sampaio ALB, Mameri .A, 2011)

3.3) Antimicrobial activity of various anti-dandruff agents.

Antimicrobial activity of banana extract, various chemical shampoos (CS-1, CS-2, CS-3) and Herbal shampoos (HS-1) were checked against Bacteria, Fungal and Malassezia spps. Which found on human healthy and affected scalp. [Table No.3.10 and Fig. No. 3.28, 3.29, 3.30]

In present study, Acetone extract of banana leave show significant antidandruff activity against Malassezia species (MI-2, MI-4, MI-5) when it was compare with sulfur powder (control). But anti-dandruff activity of acetone extract of banana were not significant than chemical antidandruff shampoo. Acetone extract of banana doesn’t show significant antibacterial and antifungal activity against Bacterial (BI-1, BI-2, BI-3, BI-4) and yeast species (YI-1, YI-2, YI3, YI-4). Chemical shampoo largely inhibit normal micro-flora of scalp than banana extract and banana extract have significant anti-dandruff activity. So banana extract can also use as antidandruff agent. In another study, acetone extract of banana failed to inhibit the growth of both Escherichia coli and Bacillus subtilis. But, the extracts might have the possibility to prevent the growth of any other bacterial culture (B.Meenashree, 2014).Antifungal activity of acetone extracts showed excellent antifungal activity against both Aspergillus terreus and Penicillium solitum. Acetone extract showed excellent antifungal activity against Aspergillus terreus, i.e. it exhibited only 1.7cm of growth in 5 days. Acetone extract showed good activity against Penicillium solitum, followed by ethanol and petroleum ether extracts. (B.Meenashree, 2014) All medicinal plants produce important secondary metabolites like terpenoids, flavonoids, polyphenols, chlorophylls, betalains etc. Among these, phenolic compounds are considered to be the chief plant constituent because of its capacity to exhibit antioxidant, anti-cancerous and anti-inflammatory properties (Nicholson and Hammerschmidt, 1992). They inhibit the free radicals which cause cell damage, by the formation of superoxidase anions and lipid peroxides (Yan Chun and Rong Liang, 1991). The total phenolic content of the acetone extract is 10.144mg/ml.

4. CONCLUSION:

Human scalp sample harbors a vast community of microbial mutualists. Dandruff occurs at all age groups. Normal micro-flora including Bacteria and Yeast of dandruff-affected scalp sample were increases as compare to healthy scalp sample. Normal micro-flora of age group (1-10 and 10-30) were increases in dandruff-affected scalp sample. But this normal micro-flora decreases in age group (30-60) and (above 60) age group because of androgen level. Malassezia count of dandruff-affected scalp sample was increases by 2 times than its original count of healthy scalp sample. It was concluded that an increases in total microbial flora was a factor in the increase of dandruff production since almost twice as many species of organisms were found in the scalp of subjects with dandruff than on the scalps of those subjects without dandruff. Malassezia furfur is the most predominant species in the dandruff scalp and Malassezia dermatis is second being isolated. The result demonstrated a significant relationship between the Malassezia species and age groups. Dandruff is linked to the balance between bacteria and fungi of the host scalp surface.

Acetone extract of banana showed anti-dandruff activity against Malassezia furfur but failed to showed antibacterial and antifungal activity. The main objective of this research is to determine its high antidandruff property, which is less harmful to the normal cells.Chemical shampoo largely inhibit normal micro-flora of scalp than banana extract and banana extract have significant anti-dandruff activity. So banana extract can also use as antidandruff agent.

Malassezia species show different response to antifungal therapy, thus correct identification of Malassezia species and antifungal susceptibility tests could facilitate selection of appropriate antifungal agents.

5. FUTURE ASPECT:

Distribution of normal micro-flora associated with dandruff and provide new avenues for the potential prevention and treatment of dandruff.

Since Banana extract have antidandruff content, it can turned into Herbal anti-dandruff shampoo.

Further experiments could be done to purification of anti-dandruff component and effect of the banana extract on human beings.

Further combination of different herbs with banana extract check for antidandruff and which will increase efficacy of herbal shampoos.

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Received on 18.07.2015 Modified on 20.08.2015

Accepted on 25.08.2015 ©A&V Publications All right reserved

Res. J. Pharmacognosy & Phytochem. 7(3): July-Sept. 2015; Page 146-162

DOI: 10.5958/0975-4385.2015.00026.6