The survey and follow-up research was executed in a planned manner in the following steps:
- Field survey
- Compilation of database and monograph (scientific literature survey of identified plants)
- Scientific evaluation (safety and efficacy) of selected medicinal plants
- Field survey
- Preparation before the field work:
It included collection of ethnobotanical data related to skin infections from literature, secondary information-regarding map, flora, land, the people, and the conservation issues in the regionwere obtained. Specific sites were selected with the help of maps before field visits.
- Formation of the multidisciplinary team:
Team for survey included dermatologist, taxonomist, pharmacist, microbiologist, chemist, biochemist, and anthropologist.
Identification of fields:
All relevant information about the flora, soil, culture, and people were obtained before visiting the field.
- Ensuring community participation:
During survey, the villagers were informed about aims and objectives of the survey in a friendly atmosphere. A dermatologist was essentially included in the survey team so that people who usually donot have easy access to doctors could discuss their diseases and medications with the doctors.
Interviewed patients, old people, and hakims to record traditional knowledge of medicinal plants, used by them to treat skin infections. It also included inquiries into types of plants collected from forests, farms, and gardens or brought to the market.
- Selection in choice of techniques:
Samples were collected from the patients by the microbiologist, which was examined in detail in various laboratories.
- Scientific evaluation:
The data recorded on questionnaire was analyzed, thoroughly. All plants and herbs used by the local people will be collected/ purchased. A full scientific evaluation of their claim has been done at International Center for Chemical and Biological Sciences (ICCBS).
- Systematic work:
The survey was carried out systematically. It included the maps of the sites to be visited, complete data of the patients, geographical information of the village surveyed, identification of the biological samples collected from patients, and scientific evaluation of the ethnobotanical information.
- Compilation of on-line database and Monograph
A database of traditional medicine used for the treatment of skin infections has been compiled, which is a frame-based on-line reference of all information on medicinal plants and extract used in the rural areas of Sindh. A monograph has been published.
This database and monograph include information about skin infections as well as plants, and herbs used in rural areas of Sindh for the treating these diseases; the data also includes herbal formulations and scientific evaluation.
- Scientific evaluation (safety and efficacy) of selected medicinal plants against skin diseases
All plants and other vegetation identified through the survey were collected, and extracted at the International Center for Chemical and Biological Sciences (ICCBS). A systematic study was conducted on the chemistry and pharmacology of extracts and compounds isolated from natural sources. Extraction, isolation, and structure of active constituents were determined by using sophisticated spectroscopic techniques, and chemical methods.
- Extraction and Isolation
Plants were collected/purchased and dried in the air. It was crushed and soaked in methanol/ethanol for one week at 25o C. The crude extract was dissolved in distilled water, and defatted with hexane. The defatted aqueous extract was further fractionated with CHCl3, EtOAc, and BuOH. These extracts were evaporated, and evaluated for their antibacterial, antifungal, and other relevant assays. The active extracts were subjected to column chromatography (CC) on silica gel, sephadex LH-20, and HPLC, and eluted with gradients 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 was carried by using UV, IR, Mass, 1- and 2-D NMR techniques, and by chemical methods.
- Bioassay and Pharmacological Evaluation
Extracts (80% EtOH/MeOH-H2O) of identified plants were prepared and dried under vacuum. These extracts were 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 was confirmed by obtaining material from an actively infected site (i.e., skin, hair, or nail), by dissolving it in a potassium hydroxide (KOH) solution, and examined the material under a microscope. Although, the KOH preparation only confirmed the presence or absence of a dermatophyte, it did not identify the responsible species. If required, then infected material was subjected to inoculation on a fungal culture medium, such as Sabouraud’s, DTM or Mycosel agar.
In circumstances, where a KOH preparation and fungal culture were negative, but a dermatophyte infection remained a strong consideration on the basis of clinical findings, then biopsy of the affected site were done. The biopsy material stained with periodic acid-Schiff (PAS), which revealed the presence of fungal elements by staining them red. The Wood’s lamp was used to detect a skin infection, produced by organism.
- Clinical Isolation
The following general methodology was employed for the clinical isolation.
Fungal isolates and inoculum preparation
Fungi and bacteria were obtained from the patients during survey. Potato dextrose agar was used to prepare homogeneous suspensions of fungi hyphal fragments. The turbidity of the supernatants was measured spectrophotometrically.
Broth microdilution antifungal susceptibility testing
The MICs of the anti-dermatophytic agents were performed with RPMI 1640 medium supplemented with L-glutamine. The pH of the medium was adjusted to pH 7.0 with 0.165 M morpholinepropanesulfonic acid buffer.
A series of double dilutions of the stock solutions of the antifungal agents (plant extracts) were prepared in 2´RPMI 1640 medium. Each dilution was 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 was inoculated and used as a growth control. The blank medium was free of drug and fungus.
The microdilution plates were incubated at 30°C for 96 h, and the endpoints were read as the MICVIS and MICCOL. Fungal growth activity was measured and calculated as a fluorescence reader.
Bacterial isolates and inoculum preparation
Samples were collected by initial examination of patient’s, such as skin culture from the infected site (skin swabs, and skin biopsy), culture of the drainage (fluid) from the infected site, blood culture, urine culture, and sputum culture. The species was identified by growing the culture on nutrient agar /nutrient broth and on other selective media (manitol salt agar etc). Characteristics and morphology was also checked by gram staining. Subsequently the biochemical tests were carried out for further confirmation. Next, the natural and synthetic compounds were screened against the particular skin infection causing bacteria by using disc diffusion method/tube dilution.
Diagnostic test of leishmaniasis (Skin split smear)
Leishmaniasis is diagnosed in the Haematology Laboratory of the ICCBS by direct visualization of the amastigotes (Leishman-Donovan bodies).
Sample taken from blood or aspirates (marrow, spleen, lymph nodes or skin lesions) were 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 stage was observed under microscope.
0.1 mL blood taken from patient’s lesions was poured in NNN biphasic medium bottle and then incubated for 72 h. Promastigotes stage of parasites was obsreved in biphasic NNN medium.
Crude extracts/pure compounds, used for treating skin infections, were subjected to cytotoxicity assay by using human neutrophils. The assay was based on the reduction of tetrazolium salt WST-1 by mitochondrial dehydrogenases of viable cells to yellow organ formation dye, which was measured spectrophotometrically.
Clinical studies on selected bioactive extracts and substances (already been evaluated for its cytotoxicity and on animal models) were 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 of extracts/drugs, used to treat or cure a specific skin infection/disease or condition were conducted. These trials were divided into three phases in order to determine effective and safe use in humans.
Phase I studies assessed the safety and side effects of drugs. About 70% of experimental drugs passed this phase of testing.
Phase II studies tested the efficacy of a drug. Most phase II studies were randomized trials where one group of patients received the experimental drug, while a second “control” group received a standard treatment or placebo. About one-third of experimental drugs successfully completed both Phase I and Phase II studies.
Phase III studies involved randomized and blind testing in several hundred to several thousand patients. This large-scale testing provided the pharmaceutical company and the FDA with a more thorough understanding of the effectiveness of the drug, the benefits, and the range of possible adverse reactions.
Systematic Flow Chart of Survey Methodolgy and Plant Screening