|Year : 2016 | Volume
| Issue : 1 | Page : 16-20
Insignificant antifungal activity of plant extracts on Malassezia furfur
Sreelatha Gopalakrishnan Lalitha, Lakshmeesha Thimappa Ramachandrappa, Soumya Krishnamurthy, Jayashree Basavaraju, Sharmila Thirumale
Department of Microbiology and Biotechnology, Jnanabharathi Campus, Bangalore University, Bengaluru, Karnataka, India
|Date of Web Publication||19-Feb-2016|
Department of Microbiology and Biotechnology, Jnanabharathi Campus, Bangalore University, Bengaluru - 560 056, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: The aim of present study was to investigate the antimalassezial potential of the selected plant extracts known to possess medicinal values. Materials and Methods: The crude extracts of five botanicals were evaluated against Malassezia furfur. The dried and pulverized plant material was extracted with water and also successively extracted with hexane, chloroform, and methanol, using the Soxhlet apparatus. The antimicrobial activity assay was done by disk diffusion and 96-well plate microdilution method. Results: The results point toward the fact that all the tested extracts extracted in solvents of different polarities exhibited no antifungal activity. Conclusion: The tested extracts did not have, or had too little, antifungal activity to be used as a promising source of novel antimicrobial substances against M. furfur.
Keywords: Disk diffusion, Malassezia furfur, microdilution, plant extracts
|How to cite this article:|
Lalitha SG, Ramachandrappa LT, Krishnamurthy S, Basavaraju J, Thirumale S. Insignificant antifungal activity of plant extracts on Malassezia furfur. J Pharm Negative Results 2016;7:16-20
|How to cite this URL:|
Lalitha SG, Ramachandrappa LT, Krishnamurthy S, Basavaraju J, Thirumale S. Insignificant antifungal activity of plant extracts on Malassezia furfur. J Pharm Negative Results [serial online] 2016 [cited 2017 Apr 23];7:16-20. Available from: http://www.pnrjournal.com/text.asp?2016/7/1/16/177055
| Introduction|| |
Pityriasis capitis, commonly known as dandruff, is a most common physiological condition causing desquamation of the skin surface due to the imbalance caused to the stratum corneum.  Seborrheic dermatitis is yet another condition that is closely related to dandruff, characterized by highly pruritic chronic inflammatory lesions on the head.  These pathologic conditions are considered to be related to three etiologic factors: Malassezia yeasts, secretions of the sebaceous glands, and susceptibility to infections in individuals. It has been generally accepted and through the literature inferred by studies, indicating a strong link to said conditions caused by Malassezia yeasts, especially Malassezia furfur and, established later, its two species forms Malassezia globosa and Malassezia restricta.  Other conditions associated with this species are pityriasis versicolor, folliculitis, atopic dermatitis, psoriasis, and confluent and reticulate papillomatosis. 
The hydrolytic enzyme lipase secreted by Malassezia species residing on the surface of the scalp and in the follicular infundibulum cleaves sebaceous triglycerides into free fatty acids and glycerol into the extracellular milieu. The Malassezia consumes necessary saturated fatty acids required for their proliferation and leave behind an increased quantity of irritating unsaturated fatty acids. The unsaturated fatty acids penetrate the stratum corneum, and in susceptible individuals, due to the nonuniform structure, breach the skin barrier function. This barrier breach induces an irritation response leading to scalp hyperproliferation, ultimately leading to pathological conditions such as dandruff and seborrheic dermatitis. 
Treatment strategies include synthetic drug-based formulations of ketoconazole, zinc pyrithione, selenium sulfide, ciclopirox, etc., which reduce visible symptoms of flaking and restore the normalcy of the skin.  In fact, most medicated formulations carry risks of side effects such as dryness of hair/skin, associated cytostasis, and eczema, and also frequent reoccurrence made therapy costlier. So, it is imperative to search for drugs that are safe, cost-effective, and eco-friendly. Thus, there was a call to return to nature and botanical remedies became a part of the green revolution.
The term "botanicals" refers to the preparations derived from herbs, spices, roots, stems, and other materials of botanical origin.  Plants that are rich in a wide array of antifungal secondary metabolites are gaining importance as a primary source of commercial medicines and drug leads, and have been used for the treatment of skin disorders for centuries.  Herbal drug technology has picked up strongly in terms of the extraction and characterization of active compounds and also the processing of herbs into medicine, considering the fact that modern medicine is not capable of providing a "cure all" solution for the pathologies mentioned above. Herbal therapy is considered as a therapeutic alternative, as a safer choice than synthetic medicines, or sometimes even as the only successful therapeutic way left to treat these disorders.  It is important to document their uses and their distinct modes of action because such information can help in obtaining maximum benefits from these resources, and this increases the possibility of their safe and efficient use in the future. Traditionally, many botanicals tend to possess anti-dandruff activity, but scientific validation is needed to justify their role in this activity. 
In this present scenario, a developing country such as India must focus on documentation of medicinal plants and development of herbal formulations to combat skin infections. The plants used in the present investigation, such as Punica granatum L. (Lythraceae), Illicium verum Hook (Schisandraceae), Nyctanthes arbor-tristis L. (Oleaceae), Thespesia populnea L. (Malvaceae), and Piper betle L. (Piperaceae) were used to treat cutaneous infections such as psoriasis, tinea infection, and eczema. ,,,, Thus, the present study was conducted with particular emphasis on aforementioned medicinal plants used traditionally against the dermatologically prevalent yeast, M. furfur.
| Materials And Methods|| |
Collection and maintenance of test microorganism
Malassezia furfur (strain no. 1374) was obtained from the Microbial Type Culture Collection and Genebank (MTCC), Chandigarh, India. The culture was maintained in Leeming-Notman (LN) agar, modified Emmons agar medium, and Sabouraud's dextrose agar (SDA) supplemented with milk. LN agar medium was used for carrying out antimicrobial studies. 
Plant materials used were selected based on their topical therapeutic properties according to the Ayurvedic system of medicine.  Authenticated plants were collected from Gandhi Krishi Vignan Kendra, Bengaluru, Karnataka, India. The voucher specimens of all the plants were maintained at the Department of Microbiology and Biotechnology, Bangalore University, Bengaluru.
Preparation of plant extracts
Fresh plant materials were blot-dried and macerated with sterile, distilled water in a blender (1:3 w/v) for 10 min. The macerate was first filtered through double-layered muslin cloth and centrifuged at 8000 rpm for 10 min. The supernatant was filtered through Whatman no. 1 filter paper and sterilized at 120°C for 30 min, and this served as the mother extract. All the extracts were concentrated in water bath and stored at 4°C for further use. 
For solvent extraction, 60 g of shade-dried plant material was pulverized, placed to fill the thimble of a Soxhlet extractor, and extracted with 250 mL of different solvents (sequentially using hexane, chloroform, and methanol). The process of extraction continued until the solvent in the siphon of the extractor become colorless. The extracts were concentrated using rotary evaporator and the dried extract was kept in the refrigerator at 4°C until further use. ,
Antimicrobial susceptibility test
As an initial step toward confirming preliminary antifungal activity, the disk diffusion assay was done to screen the antifungal activity of all the extracts. Inoculum suspension previously adjusted to 0.5 McFarland standard was swabbed evenly over the sterile LN agar medium plates set for the disk diffusion assay. Sterile 6 mm diameter disks were placed equidistantly round the margin of the Petri dish More Details. Of the test sample drug, 10 μL (25 mg mL -1 ) was dispensed on the disks. The inoculated plates were incubated at 32 ± 2°C for 48 h. Each test was performed in triplicate. After the incubation period, the antimicrobial activity was recorded as the width (diameter of inhibition zone plus diameter of the disk) of the inhibition zone. Minimum inhibitory concentrations (MICs) were determined in liquid culture by a 96-well microtiter plate assay recommended by the National Committee for Clinical Laboratory Standards (NCCLS) with minor modification.  Further, minimal yeast-cidal concentration (MYC) has been evaluated by streaking all treated samples on agar medium and assessing the concentration that inhibits complete yeast growth after incubation. 
Statistical analysis was performed using SPSS software version 16.0 (Armonk, NY, IBM Corporation). The results were expressed as mean ± (standard deviation) SD (n = 3). Univariate analysis followed by Tukey's honestly significant difference (HSD) post hoc test was applied for statistical analysis, with the level of significance set at P < 0.05. Results with P < 0.05 were considered to be statistically significant.
| Results|| |
The study examined the antimalassezial activity of selected extracts on the dermatologically prevalent yeast M. furfur using the in vitro technique, which is reliable, sensitive, and widely used. The hexane, chloroform, methanol, and water extracts of the selected plants yielded 0.48-18.15% of their dry weight. The yield was not constant with any particular solvent or plant part. Antimicrobial activity assessed in terms of zone of inhibition in mm of the ten extracts of five plants with different solvents and tested against M. furfur was recorded and can be seen in [Table 1]. In the current study, a total of ten extracts of selected plants were tested for their antifungal activity, of which all the extracts showed no activity or very little activity against M. furfur at the tested concentration of 25 mg mL -1 . Nevertheless, the highest antimicrobial activities were recorded for N. arbor-tristis in methanol and hexane, with zones of inhibition of 8.33 ± 0.57 mm and 7.73 ± 0.46 mm, respectively. In case of the hexane extract of I. verum, the zone of inhibition observed was 7.66 ± 0.57 mm and the methanol extract of P. granatum showed the inhibition zone of 7.33 ± 0.57 mm, thus manifesting limited antifungal potential against M. furfur. The extracts that showed activity in disk diffusion assay were evaluated for their MIC and MYC values by the 96-well broth dilution method and are tabulated in [Table 2]. The MIC value of the extracts that showed confined activity was 25 mg mL -1 , while the MYC value was 50 mg/mL -1 . The remaining extracts exhibited no activity.
|Table 1: Antifungal activity of different plant extracts of Malassezia furfur |
Click here to view
| Discussion|| |
Antimicrobials of plant origin are in great demand due to their widespread biological activities providing a source for the discovery of many types of effective bioactive compounds. Of these, very few successful drugs are now available for the treatment of fungal infections, especially for superficial mycoses.  Ever since the importance of the distribution of pharmacologically active principles in higher plants was understood and acknowledged, the importance of such plant-derived medicines in modern therapeutic practice has paved the way for the development of new drug leads that are safe, cost-effective, and eco-friendly. Taking this into account, the present study was conducted to assess the antifungal potential of medicinal plants, particularly to target the dandruff-causing yeast M. furfur. The present findings were in accordance to the reports of Dikshit et al., Lee et al., and Hayouni et al. ,, However, the studied extracts had relatively high MIC and MYC values and thus cannot be considered a good antifungal agent.
| Conclusion|| |
To conclude, it is imperative to emphasize the significance of more comprehensive studies that will detect new lead molecules underlying the action of crude extracts that allow for new discoveries. Essentially, the present study, which establishes the lack of significant antimalassezial activity, removes the need for any repetitive study with these extracts in the future.
The authors would like to extend their sincere thanks and appreciation to the Department of Microbiology and Biotechnology, Bangalore University for providing adequate lab facilities and the Himalaya Drug Co, Nelamangala, Bangalore for their magnanimous gesture in supporting research and providing the required plant extracts needed for the study.
Financial support and sponsorship
UGC - BSR meritorious fellowship for Sreelatha G.L.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Turner GA, Hoptroff M, Harding CR. Stratum corneum dysfunction in dandruff. Int J Cosmet Sci 2012;34:298-306.
Hay RJ. Malassezia
, dandruff and seborrhoeic dermatitis: An overview. Br J Dermatol 2011;165(Suppl 2):2-8.
DeAngelis YM, Gemmer CM, Kaczvinsky JR, Kenneally DC, Schwartz JR, Dawson TL Jr. Three etiologic facets of dandruff and seborrheic dermatitis: Malassezia
fungi, sebaceous lipids, and individual sensitivity. J Investig Dermatol Symp Proc 2005;10:295-7.
Gupta AK, Batra R, Bluhm R, Boekhout T, Dawson TL Jr. Skin diseases associated with Malassezia
species. J Am Acad Dermatol 2004;51:785-98.
Dawson TL Jr. Malassezia globosa
: Breakthrough understanding of the etiology and treatment of dandruff and seborrheic dermatitis through whole-genome analysis. J Investig Dermatol Symp Proc 2007;12:15-9.
Pierard GE, Arrese JE, Pierard-Franchimont C, DE Doncker P. Prolonged effects of antidandruff shampoos - time to recurrence of Malassezia ovalis
colonization of skin. Int J Cosmetic Sci 1997;19:111-7.
Reuter J, Merfort I, Schempp CM. Botanicals in dermatology: An evidence-based review. Am J Clin Dermatol 2010;11:247-67.
Ali-Shtayeh MS, Abu Ghdeib SI. Antifungal activity of plant extracts against dermatophytes. Mycoses 1999;42:665-72.
Joshi K, Chavan P, Warude D, Patwardhan B. Molecular markers in herbal drug technology. Curr Sci 2004;87:159-65.
Cavero RY, Akerreta S, Calvo MI. Medicinal plants used for dermatological affections in Navarra and their pharmacological validation. J Ethnopharmacol 2013;149:533-42.
Jurenka JS. Therapeutic applications of pomegranate (Punica granatum
L.): A review. Altern Med Rev 2008;13:128-44.
Matan N, Matan N. Antifungal activities of anise oil, lime oil, and tangerine oil against molds on rubberwood (Hevea brasiliensis
). Int Biodeter Biodegr 2008;62:75-8.
Rani C, Chawla S, Mangal M, Mangal AK, Khagla S, Dhawan AK. Nyctanthes arbor-tristis
Linn. (Night Jasmine): A sacred ornamental plant with immense medicinal potentials. Indian J Tradit Knowl 2012;11:427-35.
Kumar VP, Chauhan NS, Padh H, Rajani M. Search for antibacterial and antifungal agents from selected Indian medicinal plants. J Ethnopharmacol 2006;107:182-8.
Trakranrungsie N, Chatchawanchonteera A, Khunkitti W. Ethnoveterinary study for antidermatophytic activity of Piper betle
, Alpinia galanga
and Allium ascalonicum
extracts in vitro
. Res Vet Sci 2008;84:80-4.
Leeming JP, Notman FH. Improved methods for isolation and enumeration of Malassezia furfur
from human skin. J Clin Microbiol 1987;25:2017-9.
Khare CP. Indian Medicinal Plants: An Illustrated Dictionary. India: Springer; 2007. p. 325-659.
Mohana DC, Raveesha KA. Anti-fungal evaluation of some plant extracts against some plant pathogenic field and storage fungi. J Agr Sci Tech 2007;4:119-37.
Sreelatha GL, Babu UV, Sharath Kumar LM, Soumya K, Sharmila T. Investigation on biochemical characterisation and in vitro
antifungal efficacy of plant extracts on Malassezia furfur.
Int J Pharm Bio Sci 2015;6:1027-41.
Yadav RN, Agarwala M. Phytochemical analysis of some medicinal plants. J Phytol 2011;3:10-4.
National Committee for Clinical Laboratory Standards. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard. Document M-27A. National Committee for Clinical Laboratory Standards. Wayne, Pennsylvania: National Committee for Clinical Laboratory Standards; 1997.
Lakshmeesha TR, Sateesh MK, Prasad BD, Sharma SC, Kavyashree D, Chandrasekhar M, et al
. Reactivity of crystalline ZnO superstructures against fungi and bacterial pathogens: Synthesized using Nerium oleander
leaf extract. Cryst Growth Des 2014;14:4068-79.
Afolayan AJ, Grierson DS, Mbeng WO. Ethnobotanical survey of medicinal plants used in the management of skin disorders among the Xhosa communities of the Amathole District, Eastern Cape, South Africa. J Ethnopharmacol 2014;153:220-32.
Dikshit A, Tiwari AK, Mishra RK, Kamran A, Pandey A, Kumar A, et al
. Botanicals for the management of dandruff. Med Plants 2012;4:55-64.
Lee JH, Lee JS. Chemical composition and antifungal activity of plant essential oils against Malassezia furfur
. Kor J Microbiol Biotechnol 2010;38:315-21.
Hayouni EA, Miled K, Boubaker S, Bellasfar Z, Abedrabba M, Iwaski H, et al
. Hydroalcoholic extract based-ointment from Punica granatum
L. peels with enhanced in vivo
healing potential on dermal wounds. Phytomedicine 2011;18:976-84.
[Table 1], [Table 2]