Assessment of antimycotic activity of seabuckthorn (Hippophae rhamnoides) leaf exacts against common fungi associated with skin dermatitis

Aim: To study the antimycotic activity of seabuckthorn leaf extracts against common fungi associated with skin infection. Materials and Methods: A total of 31 isolates were collected from the skin cases of animals and the leaves of seabuckthorn collected from the Lahaul and Spiti valley of the Himachal Pradesh. The extracts from the leaves were extracted according to standard procedures. Antimycotic activity was examined by employing agar disc diffusion method on Roswell Park Memorial Institute medium (RPMI). Different concentrations of leaf extracts i.e. 0.50%, 2%, 3%, 4%, 5%, respectively were used. Results: The study revealed that the inhibitory effect of SBT leaf extract at 5% concentration was observed to be almost 80% in 12 hrs of reading as compared to the standard drugs used as positive control against fungal isolates. Conclusion: Plants are important source of potentially useful structures for the development of new chemotherapeutic agents. The first step towards this goal is the in vitro antimycotic activity assay. In the present study, a wide range of pathogenic microorganisms were recovered from the clinical samples, including bacteria and fungus. The seabuckthorn methanolic leaf extracts (hot and cold) were studied for their antimycotic activity against common fungal pathogens.


Introduction
for therapeutic utilization in human and Veterinary medicine, as it is reported to possess many bioactive "Antimicrobial resistance is the global problem substances notably lipids, fatty acids, vitamins (both fat that needs urgent action" [1].It is undiluted truth that soluble and water soluble), flavonoids, tannins, phenols, pain, disease and death are always associated with the steroids, 5-hydroxytryptamine etc. [2].The presence of human and animal life.The humans of early ages used these bioactive substances in the plant contributes therapeutic agents available easily to them and thus, enormously to its various properties like antibacterial, plants have been used as remedies since time immemantifungal, immunostimulant, anti-inflammatory, antiorial.The plants are therefore, man's oldest resources radiation, antioxidant, hepatoprotective, cytoprotective, of useful drugs.The history of the medicine and anticancerous, anti-artheroscle-rosis, anti-diabetic, pharmacy begins with Hippocrates, the father of anti-ulcer as well as wound healing actions [3].medicine.Medicinal herbs for the treatment of diseases In India, seabuckthorn is found in Himalayan have actively been practiced since the beginning of states of Himachal Pradesh, Ladakh (J&K), Uttranchal, human civilizations.The skin is supposed as an organ Sikkim and Arunachal Pradesh.There are about 12,000 of considerable elasticity and tensile strength that ha of seabuckthorn under thick forests, mainly in provides a barrier to the external environment.Its Ladakh.In Himachal Pradesh, seabuckthorn grows in prime function is to prevent the invasion of bacteria and Lahaul-Spiti, upper Kinnuar and Pangi in Chamba viruses and thus to maintain internal homeostasis.One district.Both H. rhamnoides and H. Salicifolia have of the major concerns in healthcare today is the been found in Chango area of upper Kinnaur [4].ineffectiveness of antibiotics towards new and re-Seabuckthorn has also been found in high altitude areas emerging diseases due to the developed resistance in of Shimla and Kullu districts."India has world's second many pathogens.Thus, there is an urgent need to largest resource of seabuckthorn.Natural products will identify novel anti-microbial molecules as leads for always be considered as one of the major sources of effective drug development.Seabuckthorn (Hippophae new drugs in the future because it offers structural species) is one among the widely known important diversity, and most are small in size.Some experts medicinal plants.The genus name Hippophae is believe that plants may well hold the secret to combat classical Latin for "shining horse," a name that was diabetes, cancer and AIDS etc.The Hiporamin a tannin given in ancient times after it was found that feeding fraction from SBT possessing a wide spectrum of the leaves to horses improved their health and made antiviral activities and mild antimicrobial activities [5].their hair shiny.The plant has got tremendous potential The present study was carried out to determine Step 4: Upper suspension of non-germinated condia/ the in vitro antimycotic activity of methanolic extracts sporangiospores/hyphal fragments mixed for 15 of seabuckthorn leaves against common fungal seconds pathogens associated with skin dermatitis [6].

Microscopic characteristics: Microscopic characteristic
The extracted liquid was subjected to rota-evaporation of fungi e.g.budding cells, hyphal structures, macroor water bath evaporation to remove the excess of conidia, microconidia and other mycotic structures methanol.The semi solid extract produced was kept in were examined.º the deep freezer at -80 C overnight.

Drug sensitivity profile of fungal isolates: Drug
Cold extraction method: 100 gm of seabuckthorn sensitivity of fungal isolates was observed by disc leaves powder was mixed with 400 ml of 100 per cent diffusion method as recommended by the NCCLS methanol and left for 24 hours in orbital shaker at 25 to (M38-A standard) [7].The fungal isolates after º 30 C at 120 rpm.Filtered the residue by using triple identification were subjected to in-vitro antifungal whatman filter paper no.1.Dried the filtrate by the susceptibility testing by disc diffusion methods.The rotator vaccume pressure for recovery of methanol.fungal isolates were grown on PDA slants for one week º The semi solid extract produced was kept in the deep at 35 C and then flooded with approximately 1ml of freezer at -80ºC overnight.sterile NSS.The resultant liquid was allowed to stand in a test tube to allow the heavier particles to settle

down. The supernatant was taken out and mixed for 10
Isolation pattern in fungal dermatitis: Microsporum min.and the opacity adjusted to 0. In this study, the extract of seabuckthorn leaves extracts were tested with a comparable concentration extract having high potential of antimycotic activity.It of antibiotics ranging 10 to 30 µg/disc.The leaf extract showed varying degrees of activities against all the was found significantly effective against M. gypseum tested dermatophytes with better antifungal activity and T. rubrum.The inhibitory effect of SBT leaf against M. gypseum and T. rubrum.The phytochemical extracts at 5 per cent concentration was observed to be studied revealed the presence of important secondary almost 50 per cent compared to the standard drugs used metabolites (alkaloids, saponin, tannin and steroids), as positive control against M. gypseum and T. rubrum thus indicating the therapeutic potential of seabuckthorn leaf.

Table -
Mucor leaves at 37 C for 3 to 5 days, there were grounded in spp.Microsporum canis, Candida spp., Rhizopus spp. the grinding machine made for the laboratory.and Absidia spp.were isolated from clinical cases of Exposure to sunlight was avoided to prevent the loss of skin and wound infections from Veterinary Clinic, active compounds [8].College of Veterinary and Animal Sciences, Palampur, Textures, topography, growth seabuckthorn leaves was kept in thimble for extraction.rate, obverse and reverse side pigmentation of colonies 400 ml of 100 per cent of methanol was used for this 1. Antifungal discs used and their concentration Plant material: The seabuckthorn leaves were collected º gypsum, Trichophyton rubrum, Aspergillus spp., º were noted.processand left for 8 hours in soxlet apparatus at 60 C.

Table - 2
. Isolation pattern in fungal dermatitis (Table3, 4 and Fig.1, 2).The cold methanolic extract was found to be most significant as compared to the hot The first step towards this goal is the in extracts of Senna alata leaf was carried on Dermatophytes vitro antimycotic activity assay[9].In the present study, which include the genera Trichophyton, Microsporum a wide range of fungal isolates were recovered from the and Epidermatophyton and their results showed that clinical samples, including dermatophytes and other the leaf exuadates and the ethanolic extract of the leaf zygomycetes.The seabuckthorn methanolic leaf of Senna alata L. Exhil: had marked antifungal effects extracts (hot and cold) were studied for their antimycotic on Microsoprum canis, Trichophyton mentagrophytes activity against common skin dermatophytes.These and Epidermatophyton spp.The ethanolic extract included 31 fungal isolates.Both SBT leaf extracts (hot showed the highest zone of inhibition on Trichyophton and cold) showed significant antimycotic activity spp.and Epidermatophyton spp.20.50 and 20.00 mm against the common fungal isolates.The antifungal zone of inhibition, respectively.activity of SBT leaf extracts (Hot and Cold) against Conclusion skinborne fungi is presented in Table-3 and 4 as zone of inhibition (in mm).Antimycotic activity of leaf º and 12.9%, respectively.Other fungi isolated were slants 7 days at 35 C Rhizopus spp.(9.67%), Candida spp.(16.13%),Step 2: Flooding of slant with approximately 1 ml of Absidia spp.(9.67%), respectively.sterile NSS and subsequently withdrawal of suspension Step 3: Heavy particles allowed settling for 3 to 5 min