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    International Center for Chemical and Biological Sciences
  • 1 Dr. Panjwani Center for Molecular Medicine and Drug Research
    International Center for Chemical and Biological Sciences
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    International Center for Chemical and Biological Sciences
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    International Center for Chemical and Biological Sciences
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    International Center for Chemical and Biological Sciences
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    International Center for Chemical and Biological Sciences
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    International Center for Chemical and Biological Sciences
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    International Center for Chemical and Biological Sciences
Survey Methodology
Survey Methodology

Survey Methodology

The research will be executed in a very planned way in following steps:

  1. Field survey
  2. Compilation of database and monograph
  3. Scientific evaluation (safety and efficacy) of selected medicinal plants

 

A.   Field survey

 

    • Preparation before the field work:

It includes collection of ethnobotanical data related to skin infections from literature, obtain secondary information-map, flora, land, the people and the conservation issues in the region. Consultation of the maps to select the specific sites and villages before visit.

    • Formation of the multidisciplinary team:

Making team for survey that includes dermatologist, taxonomist, pharmacist, microbiologist, chemist, biochemist, anthropologist.

    • Identification of fields:

All the relevant information about the flora, soil, culture, and people will be obtained before visiting the field.

    • Ensure community participation:

During survey,a friendly relationship will be established with villagers and aims and objectives of the survey will be discussed with them. Presence of dermatologist will be essential, as people usually donot have easy access to doctors, they discuss all the information of diseases and medication to doctors.

    • Interview:

Interview of patients (Questionnaire will be filled) (Annexure-I). Interview of old people, and hakims to record traditional knowledge of medicinal plants use by them to treat skin infections. It also includes inquiries into types of plants collected from the forest, farm, and gardens or brought in market.

    • Selection in choice of techniques:

Samples will be collected from the patients by microbiologist, which will be examined in detail in various laboratories.

    • Scientific Evaluation:

The data recorded on questionnaire will be analyzed, thoroughly. All plants, and herbs that will be mentioned by the local people will be collected/ purchased. A full scientific evaluation of their claim will be done at International Center for Chemical and Biological Sciences.

    • Systematic work:

The survey will be carried out systematically so that others who wish to conduct a more thorough study consult the result. This includes making the maps of the sites to be visited, recording the names and complete data and geographical information of the local people who will participate in the interview, identification of the biological samples collected from patients and scientific evaluation of the ethnobotanical information.

 

B.   Compilation of on-line database and monograph

A data-base for traditional medicine use for the treatment of skin infections will be compiled, which will be a frame-based on-line reference of all information on medicinal plants and extract used in the rural areas of Sindh. A monograph will also be published.

 This database will be formed in a way that will be convenient reference for the health professionals, scientist and general people. Technical terminology unique to folk medicine in Sindh will be described. This database and monograph will include information about the skin infections, plants, and herb use in rural areas of Sindh to treat skin diseases, herbal formulations, and scientific evaluation.

C.   Scientific evaluation (safety and efficacy) of selected medicinal plants against skin diseases

 

            All plants and other vegetative and natural species identified through the survey will be collected and extracted at the International Center for Chemical and Biological Sciences. During this project, a systematic study will be conducted on the chemistry and pharmacology of extracts and compounds isolated from natural sources. Extraction and isolation of active constituents from plants will be carried out by using a variety of chromatographic techniques such as column chromatography and HPLC techniques. The structure elucidation of active constituents will be determined by using sophisticated spectroscopic techniques, and chemical methods.

 

Extraction and Isolation

Plants will be collected/purchased and dried in air. Air-dried plant material will be crushed and soaked in methanol /ethanol for one week at 25o C. The crude extract will be dissolved in distilled water, and defatted with hexane. The defatted aqueous extract will be further fractionated with CHCl3, EtOAc and then with BuOH. These extracts will be evaporated and evaluated for their antibacterial, antifungal, and other relevant assays. Active extracts will be subjected to column chromatography (CC) on silica gel, sephadex LH-20 and HPLC and eluted withgradients of different solvents like hexane-CH2Cl2, hexane-EtOAc, CH2Cl2-EtOAc, CH2Cl2-MeOH, H2O-MeOH, etc. to yield the most active constituents from plants, and medicinal herbs. The structure elucidation of active constituents will be carried by using UV, IR, Mass, 1- and 2-D NMR techniques, and chemical methods.

 

Bioassay and Pharmacological Evaluation

All plants will be identified through survey, will be collected and extracted at the International Center for Chemical and Biological Sciences laboratories after taxonomic identification. Extracts (80% EtOH/MeOH-H2O) will be prepared and dried under vacuum. These extracts will be screened for relevant activity (reputed therapeutic activity) by using high-throughput biological and pharmacological screening protocols.

Diagnosis of fungal skin infections    

The diagnosis of a dermatophyte infection can be easily confirmed by obtaining material from an actively infected site (i.e., skin, hair, or nail), dissolving it in a potassium hydroxide (KOH) solution, and examining the material under a microscope. A KOH preparation that is positive for the presence of dermatophytes will demonstrate septatehyphae that branch at various angles in skin and nail specimens. In infected hairs, spores are generally present either on the hair surface or within the hair shaft. The presence of hyphae in hair is unusual. Although the KOH preparation confirms the presence or absence of a dermatophyte, it does not allow for identification of the responsible species. If this is necessary, infected material must be inoculated on a fungal culture medium such as Sabouraud's. DTM or Mycosel agar. A two- to four-week period is usually required for identification of the dermatophyte species.

In circumstances, where a KOH preperation and fungal culture are negative, but a dermatophyte infection remains a strong consideration on the basis of clinical findings, it may be appropriate to biopsy the affected site. The biopsy material can be stained with periodic acid-Schiff (PAS), which reveals the presence of fungal elements by staining them red. The Wood's lamp is an ultraviolet light source which is used to detect the coral red fluorescence of a skin infection called erythrasma, which is produced by the organism erythrasma bacterium. This fluorescence helps differentiate erythrasma from tineacruris. Wood's lamp illumination of some cases of Microsporum-induced tineacapitis also produces fluorescence, but the color is blue-green. However, the vast majority of tineacapitis is caused by T. tonsurans, and the infected hairs of these patients do not fluoresce under the Wood's light.

Clinical Isolation

Following general methodology will be employed for the clinical isolatetion.

Fungal isolates and inoculum preparation.

         Fungi and bacteria will be obtained directly from patients of during the surveys in various areas of Sindh, Pakistan. Potato dextrose agar will be used to prepare homogeneous suspensions of fungi hyphal fragments. The turbidity of the supernatants will be measured spectrophotometrically.

Broth microdilution antifungal susceptibility testing.

         The MICs of the anti- dermatophytic agents will be performed with RPMI 1640 medium supplemented with L-glutamine. The pH of the medium will be adjusted to pH 7.0 with 0.165 M morpholinepropanesulfonic acid buffer.

A series of doubling dilutions of the stock solutions of the antifungal agents (plant extracts) will be prepared in 2 ´RPMI 1640 medium. Each dilution will then mixed with an equal volume of a 1:50 dilution of the fungal suspension (approximately 2 ´ 104 CFU/ml) in 20% (vol/vol) Alamar Blue dye in sterile distilled water. A final volume of 200 mL of the reaction mixture contained 104 CFU of fungus per ml and the agents at concentrations ranging from 64 to 0.001 mg/mL. A drug-free medium will be inoculated and used as a growth control. The blank medium will be free of drug and fungus.

The microdilution plates will be incubated at 30°C for 96 h, and the endpoints were read as the MICVIS and MICCOL. The MICVIS is defined as the lowest concentration of an agent at which there is no visually observable growth in broth, and the MICCOL is defined as the lowest concentration of an agent that prevents the development of a red color in broth. Fungal growth activity will also be measured and calculated using fluorescence using a fluorescence reader.

 

Bacterial isolates and inoculum preparation

Collection of samples by Initial examination which is based on patient’s symptoms includes: skin culture from infected site (skin swabs, and skin biopsy), culture of the drainage (fluid) from the infected site, blood culture, urine culture and sputum culture. After that identify the species by growing the culture for 24 h at 37° C on nutrient agar /nutrient broth and also on other selective media (manitol salt agar etc), also check the characteristics and morphology by gram staining. Subsequently carry out the biochemical tests for further confirmation. Next is to screen the natural and synthetic compounds against the particular skin infection bacteria by pouring the sterile agar with organism in sterile Petri plates andallow the plates to solidify for an hour. Make wells (10 mm diameter) with the help of flamed borer on the surface of agar plates. Now Add compounds into each well after that incubate at 37° C for 24 h. Measure the zone of inhibition and from zone determine the antimicrobial activity of compounds.

Diagnostic test of leishmaniasis (Skin split smear)

Leishmaniasis is diagnosed in the haematology laboratory by direct visualization of the amastigotes (Leishman-Donovan bodies).

Smear Preparation:

 Sample taken from blood, or aspirates (marrow, spleen, lymph nodes or skin lesions) spread on a slide to make a thin smear, and stained with Leishman's or Giemsa's stain (pH 7.2) for 20 minutes. Amastigotes are seen with monocytes or, less commonly in neutrophil in peripheral blood and in macrophages in aspirates. They are small, round bodies 2-4μm in diameter with indistinct cytoplasm, a nucleus and a small rod-shaped kinetoplast. Occasionally amastigotes may be seen lying free between cells.

Blood taken from patient’s lesions, 0.1 mL blood pour in NNN biphasic medium bottle and incubate in cooled incubator for 72 h. Observed the promastigotes stage of parasites in biphasic NNN medium.

Cytotoxicity Studies

Crude extracts/pure compounds, used in skin infections,will be subjected to cytotoxicity assay. The cytotoxic activity of compounds will be studied by using human neutrophils. An in vitrospectrophotometric method will be used for this study, which measures the cell viability (%) after the incubation of test compounds with human neutrophils. The assay is based on the reduction of tetrazolium salt WST-1 by mitochondrial dehydrogenases of viable cells to yellow organ formation dye, which can be measured spectrophotometrically.

Clinical Studies

Clinical studies on selected bioactive extracts and substances (already been evaluated for its cytotoxicity and on animal models) will be carried out in dermatology department of the Jinnah Post Graduate Medical Center (Karachi) under the supervision of Prof. Dr. Azam J. Samdani as per international standards.

Clinical trials are medical research studies.  They are very scientifically controlled in order to evaluate how new drugs in development (called “investigational drugs”) treat or cure a specific skin infection/disease or condition. New drugs or treatments usually go through three phases of clinical trial testing before enough data is collected to determine that it is safe and effective for use in humans.

Phase I studies assess the safety of a drug or device. The study is designed to determine the effects of the drug or device on humans including how it is absorbed, metabolized, and excreted. This phase also investigates the side effects that occur as dosage levels are increased. About 70% of experimental drugs pass this phase of testing.

Phase II studies test the efficacy of a drug or device. Most phase II studies are randomized trials where one group of patients receives the experimental drug, while a second "control" group receives a standard treatment or placebo. About one-third of experimental drugs successfully complete both Phase I and Phase II studies.

Phase III studies involve randomized and blind testing in several hundred to several thousand patients. This large-scale testing provides the pharmaceutical company and the FDA with a more thorough understanding of the effectiveness of the drug or device, the benefits and the range of possible adverse reactions

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