[ VOLUME 5 I ISSUE 3 I JULY– SEPT 2018] E ISSN 2348 –1269, PRINT ISSN 2349-5138 “IN VITRO EVALUATION FOR ANTIDANDRUFF ACTIVITY OF SELECTED HOMOEOPATHIC MEDICINES AGAINST PITYROSPORUM OVALE”

Chetan Hanamantrao Shinde1, Vidhi Sarin2, Pashmin Kaur Anand2, Bipinraj Nirichan Kunchiraman3 & Arun Bhargav Jadhav4 1Associate Professor, Department of Homoeopathic Pharmacy, Homoeopathic Medical College & Hospital, Bharati Vidyapeeth Deemed To Be University, Pune, India. 2 Department of Microbiology, Rajiv Gandhi Institute of Information Technology and Biotechnology, Bharati Vidyapeeth Deemed To Be University, Pune, India. 3Assistant Professor, Department of Microbiology, Rajiv Gandhi Institute of Information Technology and Biotechnology, Bharati Vidyapeeth Deemed To Be University, Pune, India. 4Principal, Bharati Vidyapeeth (Deemed to be) University,, Homoeopathic Medical College and Hospital, Katraj - Dhankawadi, Pune - 411043.

Received: June 06, 2018 Accepted: July 22, 2018

ABSTRACT Recently there is an increase in the number of patients affected by recurring dandruff due to the development of antifungal resistance in pathogenic dandruff species. Current study has aimed to screen various homoeopathic medicines and their potencies for inhibition of Pityrosporum ovale. The homoeopathic medicines Azadirachta indica, Cinchona officinalis, Zincum metallicum, Iodium, Selenium, Sulphur, Acidum Benzoicum, Phosphorus, Acidum Sulphuricum and Zingiber officinale in 6C, 12C, 30C, 200C, 1M potencies were screened by tube assay method using Potato Dextrose Broth and found out that Acidum Benzoicum 6C, Cinchona officinalis 6C, Sulphur 6C, Zingiber officinale 6C, Acidum Sulphuricum 12C, Azadirachta indica 12C, Phosphorus 12C, Selenium 12C, Iodium 30C and Zincum Metallicum 200C could inhibit Pityrosporum ovale (MTCC-1374).

Keywords: Anti-dandruff, homoeopathic medicines, Acidum Sulphuricum, Acidum Benzoicum, Azadirachta indica, Cinchona officinalis, Iodium, Phosphorus, Selenium, Sulphur, Zincum Metallicum, Zingiber officinale.

INTRODUCTION Dandruff is a one of the common skin condition affecting scalp, generally caused by lipophilic yeast Malassezia (earlier known as Pityrosporum ovale) [Bergbrant IM, 1991], irrespective of region, age and sex. It is characterized by white dry flaky scales on the scalp and other intertriginous area (Rudramurthy S M et al, 2014). It is a very common condition and affects more than 50% of the world population. Since it affects the elegance and causes itching people around the world are on a lookout for effective dandruff controlling agent. In India, two species have been identified so far, M. restricta and M. furfur. There are many drugs such as chloroxine, ciclopirox, clobetasol, ketoconazole, salicylic acid, selenium sulfide, sulfur, tar and zinc pyrithione are available to treat dandruff (Ranganathan S and Mukhopadhyay T (2010), however, hardly any formulation have proven complete preventive and curative effect. Moreover, these drugs are reported to have various side effects (Tiwari1 A K, et al, 2011). As a result, scientists worldwide are looking for a new medicine or combination that can effectively control the pathogen and dandruff. Another approach is to identify medicines used in alternative medicine such as Homoeopathy. These medicines may play a significant role in treatment of dandruff without imparting any side effects. Homoeopathy offers constructive treatment of fungal infections particularly for Dandruff. Acidum Sulphuricum, Acidum Benzoicum, Azadirachta Indica, Cinchona Officinalis, Iodium, Phosphorus, Selenium, Sulphur, Zincum Metallicum and Zingiber Officinale are group of medicines used to treat symptoms similar to Pityrosporum ovale (Malassezia furfur) infection according to Complete, Murphy’s and Synthesis Homoeopathic Repertory, but there is no clear cut study regarding significant activity or mechanism of action (Zandvoort RV, 2003, Murphy R, 2014 and Schroyens F, 2014). Thus, it becomes imperative to collect, identify and validate the efficacy of Homoeopathic Medicines for management of dandruff. In the present study was aimed to screen the ability of various homeopathic medicines to inhibit pathogenic Pityrosporum ovale (Malassezia furfur) in-vitro and to study mechanism of inhibition.

MATERIALS AND METHOD Media and Chemicals - All media was procured from Hi Media Mumbai, India. All the reagents and chemicals used in this work were of AR grade and procured from MERK India. 680헒 IJRAR- International Journal of Research and Analytical Reviews Research Paper [VOLUME 5 I ISSUE 3 I JULY – SEPT 2018] e ISSN 2348 –1269, Print ISSN 2349-5138 http://ijrar.com/ Cosmos Impact Factor 4.236 Homoeopathic Medicines preparation - Homoeopathic Medicines Acidum Sulphuricum, Acidum Benzoicum, Azadirachta indica, Cinchona officinalis, Iodium, Phosphorus, Selenium, Sulphur, Zincum Metallicum and Zingiber officinale were obtained from GMP approved Standard Homoeopathic Medicines Manufacturer in 6C, 12C, 30 C, 200 C, 1M liquid potencies. Organism – Pityrosporum ovale (MTCC-1374) was obtained from Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh. It was maintained in potato dextrose agar slants containing corn oil and stored in refrigerator. For all studies dandruff cultures were grown in Potato dextrose broth containing corn oil and incubated at 25ºC for 72 h. Anti-dandruff assay Ant-dandruff activity of the medicines were determined using tube assay. Accordingly, fungal culture suspension (50µl) was inoculated on potato dextrose broth (650µl) with corn oil (100µl). Various potencies of the selected homoeopathic medicines (200µl) were added in each tube. It was followed by incubation at 25ºC for 72 h. Zinc Pyrithione (100µg/ml) was added as the positive control. Anti-fungal activity was scored by visual observation of the growth on the oil layer of media. MIC value MIC values of the selected medicines were found out using 96 well plate assay. Total volume in each well was adjusted to 100 µl with potato dextrose broth (30µl), varying potencies of homoeopathic medicines (50µl) and fungal culture (20µl, 0.5 OD). The experiment was conducted along with negative control (potato dextrose broth) and positive control (Zinc Pyrithione) and dispensing alcohol (ethanol 90%). Optical density of the culture was measured at zero hours and after 6 hrs. of incubation at 25ºC using Elisa plate reader (BIORAD) at 595 nm. The experiment was repeated three times and mean value was taken as the reading. Lysis studies for Dandruff For lysis study, the culture suspension (200µl) was mixed with 200µl of Homoeopathic medicines and incubated for 1 hr at 25ºC, after the incubation 50µl of the culture mixed with 50µl Trypan blue. The mixture was then loaded on a hemocytometer and observed under microscope to observe intact as well as lysed cells.

Result Anti-dandruff screening and MIC. All homoeopathic medicines used were showed inhibition zone around well. The activity of the medicines varied for each potencies (Table.1). The MIC of the drug varied from 6C to 200C potencies as given in the Table 2. None of the tested medicine could induce cell lysis in the culture. Table 1. Anti dandruff activity of the Homoeopathic Medicines against Pityrosporum ovale Sr. Name of Homoeopathic 6C 12C 30C 200C 1M No. Medicine Extremely Extremely 1 Selenium + + + less less Extremely 2 Cinchona officinalis + Extremely less + ++ less Extremely 3 Azadiracta indica + Extremely less +++ ++ less Extremely Extremely 4 Phosphorus + Extremely less + less less Extremely Extremely Extremely 5 Acidum Benzoicum + Extremely less less less less

6 Zingiber + Extremely less + + +

Extremely Extremely 7 Sulphur + + + less less Extremely Extremely 8 Acidum Sulphuricum + + + less less Extremely Extremely 9 Iodium + + + less less

Research Paper IJRAR- International Journal of Research and Analytical Reviews 681헒 [ VOLUME 5 I ISSUE 3 I JULY– SEPT 2018] E ISSN 2348 –1269, PRINT ISSN 2349-5138 Extremely Extremely 10 Zincum metallicum + + + less less 11 Positive Control +++ 12 Negative control Extremely less Vehicle Control 13 ++ (Dispensing alcohol) Chemical Control 14 ++ (Zinc Pyrithione)

Table 2. MIC of the Homoeopathic Medicines Sr. No. Medicine MIC value of (Homeopathic potency) 1 Azadirachta Indica 12C 2 Acidum Benzoicum 6C 3 Acidum Sulphuricum 12C 4 Cinchona officinalis 6C 5 Iodium 30C 6 Phosphorus 12C 7 Selenium 12C 8 Sulphur 6C 9 Zincum Metallicum 200C 10 Zingiber officinale 6C

Discussion The aim of the current study was to screen the ability of homoeopathic medicines to inhibit Pityrosporum ovale (Malassezia furfur). The medicines were selected as per the Complete and Murphy’s Homoeopathic Repertory. The results of the screening study it was clear that selected medicines could inhibit Pityrosporum ovale (Malassezia furfur) in vitro, since they showed inhibition in tube assay. MIC value varied from 6C to 200C potencies. Pityrosporum ovale is a normal human skin flora associated with various skin conditions such as pityriasis versicolor, pityrosporum folliculitis, seborrhoeic dermatitis (dandruff) and some forms of atopic dermatitis. Formation of dandruff is a complex mechanism and the role lipophilic yeast such as Pityrosporum was widely accepted (Balows A, 1998 and Saint-Leger 1988). There are many anti-dandruff formulation available in the open market, most of them act as anti-fungals and can decrease the number of Pityrosporum yeast, However, consistent use of anti-dandruff treatment lose efficacy which might develop a resistant strain (https://www.adorebeauty.com). Present study shows the effectiveness of homoeopathic medicines in controlling the Pityrosporum ovale in vitro. This study also indicates that these medicines can inhibit the fungus even at very low concentration nullifying the speculation about possible placebo effect in homoeopathic medicines.

Conclusion The present study reveals that homoeopathic medicines can effectively inhibit Pityrosporum ovale (Malassezia furfur). The tested medicines at various potencies can be an effective alternative to currently used antidandruff drugs. However, it needs further investigations to be performed to understand the molecular events involving the inhibition of Pityrosporum ovale by homoeopathic medicines.

Acknowledgement The authors are grateful to Bharati Vidyapeeth (Deemed To Be University) for providing facility to carry out the study.

References – 1. Bergbrant I M (1991) Seborrhoeic dermatitis and Pityrosporum ovale: cultural, immunological and clinical studies, Acta Derm Venereol Suppl (Stockh). 167:1-36. 2. Rudramurthy S M, Honnavar P, Dogra S, Yegneswaran P P, Handa S, & Chakrabarti A (2014), Association of Malassezia species with dandruff, Indian J Med Res 139:431-437. 3. Ranganathan S and Mukhopadhyay T (2010), Dandruff: the most commercially exploited skin disease, Indian J Dermatol. 55(2): 130–134.

682헒 IJRAR- International Journal of Research and Analytical Reviews Research Paper [VOLUME 5 I ISSUE 3 I JULY – SEPT 2018] e ISSN 2348 –1269, Print ISSN 2349-5138 http://ijrar.com/ Cosmos Impact Factor 4.236 4. Tiwari A K, Mishra R K, Awadhesh K, Srivastava S, Dikshit A, Pandey A, Bajaj K, (2011) A comparative novel method of antifungal susceptibility for Malassezia furfur and modification of culture medium by adding lipid supplement, Journal of Phytology, 3(3): 44-52. 5. Zandvoort RV, Complete Repertory, English (version 2003). Radar 10.0 6. Murphy R (2014), Homoeopathic Medical Repertory, A Modern Alphabetical and Practical Repertory, Third Revised Edition. New Delhi (INDIA): B Jain Publication; 389. 7. Schroyens F (2014), Augmented Clinical Synthesis, Repertorium Homeopathicum Syntheticum, Edition 9.1. New Delhi (INDIA): B Jain Publication; 650, 1867. 8. Balows A, Sussman M. Collier (1998). 9th ed. Vol 4. New York: Oxford University Press, Inc; Medical Mycology, Microbiology and Microbial Infections; pp. 201–11. 9. Saint-Leger D, Kligman AM, Stoudemyer TJ (1988). The role of the resident micro flora in the pathogenesis of dandruff. J Soc Cosmet Chem. 40:109–17. 10. https://www.adorebeauty.com.au/scalp-care/guide/anti-dandruff-effectiveness.

Research Paper IJRAR- International Journal of Research and Analytical Reviews 683헒